Attaching nucleic acids to platinum electrodes

Abstract

The present invention relates to a method for attaching a platinum electrode to a nucleic acid, comprising the steps of attaching an ethylenediamine molecule to the electrode (S1), which comprises the electrolytic oxidation of the primary amine of the ethylenediamine molecule by cyclic voltammetry, attaching a sulfo-SMCC molecule of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester to the ethylenediamine molecule (S2), attaching a nucleic acid to the sulfo-SMCC molecule (S3), the nucleic acid having been previously modified to comprise a thiol functional group, and during steps (S1), (S2) and (S3) the electrode is contacted with an aqueous solution, i.e. with a solution in which the solvent is water.

Description

[Technical field]

[0001] The present invention relates to the attachment of nucleic acids to platinum electrodes, and in particular to the attachment of nucleic acids to platinum microelectrodes in aqueous solutions.

[0002] The present invention also relates to platinum electrodes and therefore to the functionalization and use of such electrodes for the detection of target nucleic acids. [Background technology]

[0003] Gene detection by electrochemical techniques is known. These techniques generally use a gold electrode to which a self-assembled monolayer (also known as SAM) is attached. For the attachment of this monolayer, traditionally thiol functional groups are used, since they comprise a sulfur atom that has a strong affinity with the gold of the electrode. It is known that the stability of such self-assembled monolayers is not sufficient due to chemical desorption processes. This is mainly due to the weak nature of the gold-sulfur "Au-S" bond, which has a very weak bond energy of ∼40 kcal / mol compared to the strong carbon-carbon "C-C" type covalent bond of about 85 kcal / mol. Furthermore, the Au-S bond may be affected by its susceptibility to oxidation (change in degree of oxidation) or susceptibility when the gold electrode is polarized. Furthermore, the gold oxidation process in aqueous solution is very fast, which tends to reduce the stability of such electrodes used in aqueous solution.

[0004] Therefore, there is a need for an electrochemical sensing device with good electrode stability. Summary of the Invention

[0005] The aim of the present invention is to propose an electrochemical detection device which has better electrode stability compared to the prior art.

[0006] The object is to provide a method for attaching nucleic acids to a platinum electrode, comprising the following steps: Step (S1) of attaching ethylenediamine molecules to an electrode, which step (S1) comprises electrooxidation of the primary amines of the ethylenediamine molecules by cyclic voltammetry; (S2) attaching a sulfo-SMCC molecule of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester to an ethylenediamine molecule; This is achieved in the context of the present invention by a method comprising a step (S3) of attaching a nucleic acid to a sulfo-SMCC molecule, the nucleic acid having been previously modified to comprise a thiol functional group, and in which during steps (S1), (S2) and (S3) the electrode is contacted with an aqueous solution, i.e. with a solution in which the solvent is water.

[0007] This method makes it possible to functionalize platinum electrodes, i.e. materials in the electrodes that have better electrochemical stability than gold. Electrochemical detection devices based on such electrodes solve the problems mentioned above.

[0008] Such methods are advantageously and optionally supplemented by incorporating the following different characteristics, either alone or in combination: The aqueous solution is a physiological solution; the electrode is a microelectrode disposed at least partially in the microfluidic channel; Step (S3) is followed by a stabilization step (S4) in which the electrode is placed in a physiological solution for a period of between 20 and 40 minutes, the solution having a NaCl concentration comprised between 0.4 and 0.6 molar.

[0009] The invention also relates to a platinum electrode for detecting a target nucleic acid comprising an ethylenediamine molecule attached to the electrode, a sulfo-SMCC molecule of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester being attached to the ethylenediamine molecule by electrooxidation of a primary amine of the ethylenediamine molecule by cyclic voltammetry, and a probe nucleic acid being attached to the sulfo-SMCC molecule, the probe nucleic acid comprising a thiol functional group, the probe nucleic acid being complementary to the target nucleic acid.

[0010] The present invention further relates to an apparatus for detecting a target nucleic acid comprising the above-mentioned platinum electrode, a counter electrode, and an electrical measurement system electrically connected to the platinum electrode and the counter electrode.

[0011] Such a device is advantageously and optionally supplemented by a microfluidic channel, the platinum electrode being a microelectrode, and the platinum electrode and the counter electrode being at least partially located in the microfluidic channel.

[0012] The present invention also relates to a system for detecting a target nucleic acid comprising a plurality of devices as presented above, the system comprising an inlet configured to receive a solution to be analyzed, the inlet being connected to each platinum electrode of each device.

[0013] Such a system may advantageously and optionally be configured such that two of the multiple devices are configured to detect two different target nucleic acids.

[0014] Finally, the present invention relates to a method for detecting a target nucleic acid in a solution to be analyzed, the method comprising the following steps: Step (E1) of providing a working electrode, the working electrode being a platinum electrode as set forth above; providing a counter electrode (E2); (E3) electrically energizing the working electrode and the counter electrode to maintain a working voltage between the working electrode and the counter electrode; A step (E4) of contacting the working electrode and the counter electrode with the solution to be analyzed, A step (E5) of determining the intensity of the measured current between the working electrode and the counter electrode, determining the presence of a target nucleic acid in the solution to be analyzed based on the measured intensity (E6), It concerns the method.

[0015] The method advantageously and optionally further comprises: The following sub-steps: A first substep (SE1) of providing two reference solutions having different concentrations of the target nucleic acid; a second substep (SE2) of measuring a reference intensity between the working electrode and the counter electrode for each reference solution, the electrodes being in contact with the reference solutions and electrically energized to maintain a working voltage between the working electrode and the counter electrode; A third substep (SE3) of determining the resulting correlation of the current intensity between the working electrode and the counter electrode with the target nucleic acid concentration in the solution to be analyzed. A calibration step comprising: The method comprises determining the target nucleic acid concentration in the solution being analyzed from the measured intensities using the correspondence relationship; The target nucleic acid is a nucleic acid fragment of a pathogen. [Brief description of the drawings]

[0016] Other characteristics and advantages of the invention will appear from the following description, which is given by way of example only and is non-limiting, and which should be read in conjunction with the accompanying drawings, in which: [Figure 1] 1 to 3 are schematic diagrams illustrating steps of a method for attaching nucleic acid to an electrode according to one embodiment of the present invention. [Diagram 2] 1 to 3 are schematic diagrams illustrating steps of a method for attaching nucleic acid to an electrode according to one embodiment of the present invention. [Diagram 3] 1 to 3 are schematic diagrams illustrating steps of a method for attaching nucleic acid to an electrode according to one embodiment of the present invention. [Figure 4]FIG. 4 is a schematic diagram of a hybridization reaction of a target nucleic acid. [Diagram 5] FIG. 5 is a schematic diagram of an electrochemical assay for the detection of a target nucleic acid. [Figure 6] FIG. 6 is a schematic diagram of a nucleic acid detection device. [Figure 7] FIG. 7 shows a detail of the apparatus shown in FIG. [Figure 8] 8-9 are schematic diagrams of electrochemical measurements for detection of target nucleic acids. [Figure 9] 8-9 are schematic diagrams of electrochemical measurements for detection of target nucleic acids. [Figure 10] FIG. 10 is a schematic diagram of a target nucleic acid detection system according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Method for attaching nucleic acids to platinum electrodes A method according to one embodiment of the present invention for attaching nucleic acids to a platinum electrode is presented in connection with FIGS.

[0018] First, a platinum electrode 20 is provided, which in the context of this description is also referred to as the "working electrode." In an optional preliminary step, this electrode can be characterized to assess its surface condition and electrochemical properties.

[0019] For this purpose, two types of measurements can be implemented: cyclic voltammetry measurements and chronoamperometry measurements.

[0020] In these two measurements, a platinum electrode 20 is placed close to the counter electrode and a liquid comprising iron (III) ions, for example potassium ferricyanide, is placed in contact with the platinum electrode 20 and the counter electrode. For example, an aqueous solution of potassium ferricyanide can be used. The solution comprising potassium ferricyanide also comprises ferrocyanide ions, Fe(II), and the ferricyanide / ferrocyanide mixture may or may not be equimolar (1 / 1). The concentration of the solution comprising potassium ferricyanide is selected from a concentration equal to or greater than 3 mmol / liter and equal to or less than 30 mmol / liter, for example equal to 20 mmol / liter. The solution comprising iron (III) ions can be flowed over the electrode at a selected flow rate equal to or greater than 0.1 μL / sec and equal to or less than 1 μL / sec, preferably equal to 0.5 μL / sec.

[0021] An aqueous solution is defined as a solution in which the solvent is water, i.e. the majority component of the solution is water. A salt, whose ions ensure ionic conductivity, is generally dissolved in the solution. This combination (solution + salt) is generally called the supporting electrolyte.

[0022] A voltage is applied between the platinum working electrode 20 and the counter electrode and the current between them is measured.

[0023] Cyclic voltammetry involves scanning the voltage from -200 millivolts to +200 millivolts at a scan rate of 10 millivolts / second and measuring the current during the scan.

[0024] Chronoamperometry consists of holding the voltage constant, for example at -200 mVolt, and measuring the current between the electrodes during a monitoring period lasting, for example, 2 minutes.

[0025] It should be noted that electrochemical impedance spectroscopy (EIS) measurements can be performed in constant voltage mode (also known as "constant voltage electrochemical impedance spectroscopy" abbreviated as PEIS) or constant current mode (also known as "constant current electrochemical impedance spectroscopy" abbreviated as GEIS). Typically, PEIS measurements are performed at a scan frequency of 1.0 MHz to 100 mHz to evaluate the electrochemical properties of the electrodes. In this case, a zero potential difference is continuously applied between the platinum electrode and the counter electrode, and an AC voltage of 10 millivolts is also continuously applied, and the AC voltage is scanned at a frequency of 1.0 MHz to 100 mHz.

[0026] In step S1, as shown in Figure 1, molecules of ethylenediamine (hereinafter abbreviated as EDA) 22 are attached to an electrode 20. An aqueous solution having a certain concentration of EDA is brought into contact with the platinum electrode 20. In particular, the aqueous solution can be a physiological solution.

[0027] A physiological solution is defined as an aqueous solution having a sodium chloride concentration of greater than or equal to 0.4 mol / liter and less than or equal to 0.6 mol / liter, for example a concentration of 0.5 mol / liter.

[0028] The EDA concentration may be greater than or equal to 1 mmol / liter and less than or equal to 10 mmol / liter, for example a concentration of 2 mmol / liter.

[0029] During attachment, the primary amine NH2 of the EDA molecule reacts with platinum to form a platinum + EDA product 24.

[0030] The attachment reaction comprises the electro-oxidation of the primary amine of the EDA molecule, which is monitored by cyclic voltammetry: the voltage can be swept from 0 Volts to +1200 mVolts for several cycles, for example 10 cycles.

[0031] Electrooxidation consists of the covalent grafting of a layer of organic molecules as a result of an electrochemical reaction that oxidizes or reduces certain chemical groups on the surface of an electrode. Here, electrooxidation of a primary amine on a platinum electrode allows the electrografting of ethylenediamine to be carried out. In this way, a covalent bond is created between the nitrogen of ethylenediamine and the surface of the platinum electrode.

[0032] During step S1, several EDA molecules can be attached to the electrode 20, resulting in the formation of a self-assembled layer of EDA molecules on the surface of the electrode.

[0033] At the end of step S1, a step of characterizing the platinum+EDA electrode 24 can be performed, using one of the measurements previously described, cyclic voltammetry and / or chronoamperometry.

[0034] In step S2, a sulfo-SMCC molecule 26 of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester is attached to an ethylenediamine molecule 22 previously attached to a platinum electrode 20, as shown in FIG.

[0035] More precisely, the sulfo-SMCC molecule 26 is attached to the distal end of the EDA molecule 22 , which is opposite the proximal end of the EDA molecule that is attached to the platinum electrode 20 .

[0036] The sulfo-SMCC molecule 26 has an N-hydroxysuccinimide functional group (commonly abbreviated as "NHS") that reacts with the second amine of the EDA molecule to form the platinum+EDA+sulfo-SMCC 28 product. The second amine of the EDA molecule forms the distal end.

[0037] In this step S2, an aqueous solution having a concentration of sulfo-SMCC molecules is brought into contact with the platinum+EDA electrode 24. In particular, the aqueous solution can be a physiological solution.

[0038] The concentration of the SMCC sulfomolecule may be greater than or equal to 1 mmol / liter and less than or equal to 100 mmol / liter, for example, a concentration of 10 mmol / liter.

[0039] This attachment reaction can be carried out statically, in particular at a pH of 7.5 or more and 9 or less, for a period of 45 minutes or more and 1 hour 15 minutes or less, preferably equal to 1 hour. A static reaction in this context means that a volume of a solution, a single block and a fixed amount, comprising sulfo-SMCC molecules is brought into contact with a platinum electrode. In particular, there is no flow of solution flowing over the platinum electrode.

[0040] In step S2, several sulfo-SMCC molecules can each be attached to the EDA molecules attached to the electrode 20. A self-assembled monolayer of the aforementioned EDA molecules is completed with the sulfo-SMCC molecules.

[0041] The EDA+sulfo-SMCC assembly attached onto the electrode is referred to as the linker, referenced 30 in FIG.

[0042] At the end of step S2, a step of characterizing the Pt+EDA+Sulfo-SMCC28 electrode can be performed using one of the measurements described above, cyclic voltammetry and / or chronoamperometry and / or PEIS.

[0043] In step S 3 shown in FIG. 3, a nucleic acid 32 is attached to a sulfo-SMCC molecule 26 previously attached to a platinum electrode 20 via an EDA molecule 22 .

[0044] More specifically, nucleic acid 32 is attached to a distal end of sulfo-SMCC molecule 26 opposite a proximal end of sulfo-SMCC molecule 26 , which in turn is attached to a distal end of EDA molecule 22 .

[0045] The nucleic acid 32 is pre-modified to comprise at one of its moieties a thiol functional group, i.e. an -SH functional group comprising a sulfur atom S and a hydrogen atom H. It is the thiol functional group that attaches the nucleic acid to the sulfo-SMCC molecule 26, more precisely to the maleimide functional group of the sulfo-SMCC molecule 26.

[0046] In this step S3, an aqueous solution having a nucleic acid concentration is brought into contact with a platinum+EDA+sulfo-SMCC electrode 28. In particular, the aqueous solution can be a physiological solution.

[0047] The concentration of the nucleic acid molecule may be greater than or equal to 0.1 micromoles / liter and less than or equal to 10 micromoles / liter, for example a concentration of 1 micromoles / liter.

[0048] This attachment reaction of step S3 can in particular be carried out statically for a period of at least 1 hour 45 minutes and at most 2 hours 15 minutes, preferably equal to 2 hours.

[0049] Once the nucleic acid 32 is attached to the "linker" 30, a platinum+EDA+sulfo-SMCC+nucleic acid product 34 is formed.

[0050] The attachment reaction of step S3 can in particular be carried out statically, ie without a flow of nucleic acid solution, or quasi-statically, ie with a flow rate that is almost zero, for example ≧0.01 μL / s and ≦0.05 μL / s.

[0051] During step S3, some nucleic acid can be attached to the sulfo-SMCC molecules 26 that are attached to the platinum electrode 20 via the EDA molecules 22. The self-assembled monolayer of EDA molecules supplemented with sulfo-SMCC molecules described above is then supplemented with nucleic acid.

[0052] Step S3 can be followed by an optional stabilization step S4 during which the electrodes are placed in the physiological solution for a period of between 20 and 40 minutes, ideally 30 minutes.

[0053] At the end of step S3, and if appropriate in step S4, a step of characterizing the Pt+EDA+Sulfo-SMCC+Nucleic Acid electrode 34 can be performed using one of the measurements described above, cyclic voltammetry and / or chronoamperometry and / or PEIS.

[0054] A method for attaching nucleic acids to a presented platinum electrode can use a "linker" attached to the platinum electrode, allowing the nucleic acid to be attached via a thiol functional group.

[0055] The use of thiol functional groups to directly attach nucleic acids to platinum electrodes is not sufficient, as the affinity between platinum and this functional group is low.

[0056] All steps of the method described herein are carried out in aqueous solutions, which allow for handling of nucleic acids and attachment of nucleic acids to electrodes, and in particular, no organic solvents are used, which can degrade nucleic acids and prevent them from attaching to platinum electrodes.

[0057] Platinum electrodes for detecting target nucleic acids Detection of the target nucleic acid can use platinum electrodes to attach strands of complementary probe nucleic acid to the target nucleic acid.

[0058] To this end, an object of the invention is also a platinum electrode having an ethylenediamine molecule 22 attached to the electrode, a sulfo-SMCC molecule 26 of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester attached to the ethylenediamine molecule 22, and a probe nucleic acid 32 attached to the sulfo-SMCC molecule 26, the probe nucleic acid 32 comprising a thiol functional group, and the probe nucleic acid 32 being complementary to a target nucleic acid 36.

[0059] Such electrodes can be made by the nucleic acid attachment methods presented previously where the attached nucleic acid is a probe nucleic acid complementary to the target nucleic acid 36 .

[0060] The platinum electrode may in particular be a microelectrode.

[0061] A microelectrode is defined herein as an electrode having a dimension of at least 1000 μm or less.

[0062] The above described deposition method can be carried out on various types of platinum electrodes, especially microelectrodes.

[0063] The microelectrodes can be fabricated by photolithography.

[0064] In particular, platinum can be deposited on a glass substrate in the form of tracks obtained after a cleanroom deposition and lithography process, the electrodes consisting of said platinum tracks.

[0065] The microelectrode has a length in a first direction and a width in a second direction perpendicular to the first direction.

[0066] The dimensions of the working microelectrode may be, for example, 30 μm wide and 300 μm long.

[0067] In the electrode characterization process described above in conjunction with the accompanying method, a counter electrode can be placed close to the platinum electrode, which can be fabricated simultaneously with the platinum electrode by deposition on a glass substrate by lithography in a clean room.

[0068] The dimensions of the counter electrode are chosen to be larger than those of the working electrode, for example 2000 μm wide and 300 μm long. Such dimensions then make the counter electrode a microelectrode.

[0069] Such microelectrodes may be disposed at least partially within a microfluidic channel.

[0070] A microfluidic channel allows for the flow of fluid and has a dimension in a plane transverse to the general direction of flow of the fluid that is greater than or equal to 100 μm and less than or equal to 500 μm.

[0071] The channel has, in a plane transverse to the general direction of flow of the fluid, a width in the plane of the electrodes and a height perpendicular to the plane of the electrodes.

[0072] It is possible to carry out all of the steps of the method in which the nucleic acid is deposited on a microelectrode that is at least partially disposed in a microfluidic channel.

[0073] Preferably, the channel is placed on the microelectrode so that the flow direction is perpendicular to the length of the electrode. When the length of the electrode is greater than the width of the microfluidic channel, the effective length of the electrode, i.e. the length seen by the liquid transported in the channel, is fixed by the width of the channel. For example, in a channel width of 300 μm, the effective length of the working electrode is 300 μm for a width of 30 μm, and the effective length of the counter electrode is 300 μm for a width of 2000 μm.

[0074] More precisely, the fact that all the steps of the deposition method are carried out in aqueous solution makes it possible to operate in microfluidic channels, in particular without the use of organic solvents, which would otherwise hinder operations in microfluidic channels and which would impair the structure of the latter and the connections essential for the liquid supply.

[0075] Processing in microfluidic channels allows controllable flow regimes, especially convection. This has the advantage that certain reactions can be obtained more quickly by reducing the diffusion time to the electrode (which occurs in the thickness of the diffusion layer created on the surface of the electrode). Target nucleic acids present in the liquid flowing through the channel collide more quickly with the surface of the electrode and initiate a hybridization reaction with the probe nucleic acid attached to the platinum electrode.

[0076] To at least partially position the microelectrodes in a microfluidic channel, the microfluidic channel can be manufactured in the PDMS resin, for example in the form of an open groove on the surface of the PDMS resin part, the groove running in the elongation direction of the microfluidic channel.

[0077] In a plane perpendicular to this direction of extension, the groove can have a rectangular portion, in which case the groove is open so that the PDMS resin defines only three rectangular sides of the rectangular portion.

[0078] The PDMS resin is then bonded to a substrate carrying the microelectrodes, such as a glass substrate, so that the substrate is in close proximity to the microfluidic channels.

[0079] Where the groove has a rectangular portion, the microfluidic channel has a rectangular portion with three sides formed by the PDMS and the remaining side formed by the substrate.

[0080] The PDMS resin is aligned so that the electrode tracks reside partially within the microfluidic channels.

[0081] Polymeric materials other than PDMS can be used for the microfluidic channels, such as PMMA, PMP, PVDF, COC, PET, or block copolymers.

[0082] Other manufacturing techniques such as chemical etching, molding, embossing, or 3-D printing onto glass can be used to fabricate the microchannels.

[0083] To carry out the different steps of the above-mentioned methods, different solutions providing reagents can be inserted into the microfluidic channels.

[0084] In the electrode characterization step, a counter electrode can be previously placed in the microfluidic channel in close proximity to the platinum electrode.

[0085] Apparatus for detecting a target nucleic acid The platinum electrode for detecting the target nucleic acid presented above may in particular be provided in an apparatus for detecting the target nucleic acid, which further comprises a counter electrode and an electrical measurement system electrically connected to the platinum electrode and the counter electrode.

[0086] The platinum electrode to which the probe nucleic acid is attached is hereinafter referred to as the working electrode.

[0087] The counter electrode is placed sufficiently close to the working electrode so that a potential difference can be applied between them and the current between them can be measured.

[0088] To this end, the device comprises an electrical measurement system configured to apply a potential difference between the electrodes, measure this potential difference and measure the current between the electrodes.

[0089] The device is constructed so that the solution to be analyzed can be contacted simultaneously with the working and counter electrodes.

[0090] As shown in FIG. 4, when the solution to be analyzed contains a target nucleic acid, a hybridization reaction between the target nucleic acid and the probe nucleic acid takes place on the working electrode.

[0091] The working electrode comprises a functionalized assembly 34, platinum+EDA+sulfo-SMCC+nucleic acid electrode, as shown in Figure 4. The functionalized assembly 34 comprises a probe nucleic acid complementary to a target nucleic acid 36.

[0092] A hybridization reaction occurs when the functionalized assembly 34 is contacted with a solution containing the target nucleic acid 36. The target nucleic acid 36 and the probe nucleic acid 32 of the functionalized assembly 34 hybridize.

[0093] Thus, a hybrid 38 "probe nucleic acid 32 + target nucleic acid 36" appears on the platinum electrode. The electrochemical properties of the electrode are altered by this hybridization, so that the presence of the target nucleic acid 36 can be detected.

[0094] If the platinum electrode comprises several probe nucleic acids, the presence of the target nucleic acid 36 creates one or more hybrids 38 "probe nucleic acid 32 + target nucleic acid 36" on the platinum electrode. The other probe nucleic acids that wish to undergo hybridization remain as non-hybridized groups 40. The electrochemical properties of the electrode are modified by the hybridization, depending on the proportion of hybrids 38 and non-hybridized groups 40. These modifications make it possible to estimate the concentration of the target nucleic acid in the solution being analyzed.

[0095] This hybridization reaction is preferably carried out statically for a period of not less than 15 minutes and not more than 1 hour, preferably equal to 30 minutes.

[0096] The modification of the electrochemical properties of the electrode can be demonstrated by an electrical measurement system.

[0097] The graph shown in FIG. 5 illustrates such a modification.

[0098] The graph was obtained experimentally using a platinum electrode for the detection of a target nucleic acid as described above, more precisely an electrode with a self-assembled monolayer of EDA molecules supplemented with sulfo-SMCC molecules and a probe nucleic acid complementary to the target nucleic acid.

[0099] The electrodes are used in the detection device described, namely further comprising a counter electrode and an electrical measurement system electrically connected to the platinum electrode and the counter electrode.

[0100] 10 each -18 , 10 -16 , 10 -14 , 10 -12 , 10 -10 , 10 -8 and 10 -6 Different standard solutions with equal concentrations of target nucleic acid in moles / liter were used.

[0101] A neutral reference solution containing no target nucleic acid was also used.

[0102] For each solution, the following electrochemical measurements were carried out: the potential difference (VT-VCE) is varied between the potential difference VT of the working electrode and the potential difference VCE of the counter electrode to measure the electric field strength between these electrodes.

[0103] The potential difference (VT-VCE) is plotted against the abscissa axis in FIG. 5, and the electric field strength is plotted on the ordinate axis.

[0104] The potential difference (VT-VCE) is swept from -200 millivolts to +200 millivolts.

[0105] First, the electrodes are contacted with a reference solution and the electrochemical measurements described above and shown by curve 50 are carried out.

[0106] Curve 50 appears in FIG. 5 as a quasi-linear curve.

[0107] Other electrochemical measurements using other reference solutions are then performed on the same instrument.

[0108] Each of the curves 52, 54, 56, 58, 60, 62 and 64 corresponds to a standard solution of 10 -18 , 10 -16 , 10 -14 , 10 -12 , 10- 10 , 10 -8 and 10 -6 This represents the case where the target nucleic acid has a concentration equal to moles / liter.

[0109] After each electrochemical measurement, the electrodes are rinsed with physiological solution, ie, a neutral reference solution.

[0110] To ensure that the working electrode surface is modified, as required, with the SAM on the one hand and with the probe and target nucleic acid on the other hand, measurements with Fe(II) / Fe(III) are carried out.

[0111] The electrodes are rinsed again with physiological solution and electrochemical measurements are performed with a new reference solution having a non-zero target nucleic acid concentration.

[0112] It should be noted that the measurements are performed in order of increasing nucleic acid concentration of the reference solutions.

[0113] It is also possible to change the two electrode pairs (working and counter electrodes) between each measurement.

[0114] Curves 52, 54, 56, 58, 60 and 62 also appear similar to the linear curve of Figure 5, with the slope of the curve becoming less and less as the concentration of target nucleic acid increases. In other words, the electrochemical properties of the electrode are altered in the presence of target nucleic acid, with the alteration becoming much more pronounced at higher target nucleic acid concentrations.

[0115] The previously presented devices for detecting target nucleic acids make it possible to detect the presence or absence of nucleic acid in a solution in contact with the electrodes of the device.

[0116] This experiment was performed with probe DNA sequences to detect the coding RNA targets (E, N, RdRp) and negative control sequences. Table 1 shows the different sequences used.

[0117] [Table 1]

[0118] Optionally, the device further comprises a microfluidic channel, wherein the platinum electrode and the counter electrode are microelectrodes, and wherein the platinum electrode and the counter electrode are at least partially located in the microfluidic channel.

[0119] The device is configured such that during operation, fluid can flow through the microfluidic channel and contact the working and counter electrodes.

[0120] FIG. 6 illustrates a schematic example of an apparatus 100 for detecting a target nucleic acid.

[0121] The device 100 comprises a glass substrate 1 on which platinum has been deposited in the form of two metal tracks 2 and 3, comprising a working track 2 and a reference track 3. This deposition can in particular be carried out by the previously described deposition process and lithography in a clean room.

[0122] This working track 2 has a metallic end called the working electrode 5. It is to this working electrode 5 that the probe nucleic acid is attached.

[0123] The reference track 3 has a metallic termination called the counter electrode 6 .

[0124] The working electrode 5 and the counter electrode 6 are visible in FIG. 7, which is an enlargement of area A of FIG.

[0125] A rectangular section of microfluidic channel 4 is formed by bonding PDMS resin to a glass substrate 1. The microfluidic channel 4 is positioned relative to the substrate such that it incorporates part of the metal terminations of the metal tracks 2 and 3.

[0126] The dimensions of the microfluidic channel 4, the working electrode 5 and the counter electrode 6 are adjusted such that only a part of the working electrode 5 and only a part of the counter electrode 6 are contained within the microfluidic channel.

[0127] Furthermore, the surface of the counter electrode 6 in the channel 4 is much larger than the surface of the working electrode 5 in the channel 4. This allows the counter electrode 6 to be used not only as a counter electrode but also as a false reference.

[0128] The microfluidic channel 4 has an inlet 7 and an outlet 8. Fluid introduced at the inlet 7 passes through the microchannel 4 to the outlet 8. During this traverse the fluid comes into contact with the working electrode 5 and the counter electrode 6. The fluid does not come into contact with the rest of the metal tracks 2 and 3.

[0129] The apparatus 100 includes an electrical measurement system configured to apply a potential difference between the electrodes, measure this potential difference, and measure the current between the electrodes. The electrical measurement system may in particular be made up of the following elements: a device 9 of the potentiostatic type, consisting of a potentiostatic meter, the device 9 having three outlets, namely a working outlet 10, a reference outlet 11 and a counter electrode outlet 12; a working connector 13 connecting the working outlet 10 to the metal track 2, a reference connector 14 connecting the reference outlet 11 and the counter electrode outlet 12 to the reference track 3 .

[0130] Detection system with multiple detection devices Another object of the present invention is a target nucleic acid detection system comprising a plurality of the nucleic acid detection devices just presented.

[0131] The system comprises an inlet configured to receive a solution to be analyzed, the inlet being connected to each platinum electrode of each device, in other words, the solution to be analyzed introduced into the inlet of the system enters each device and travels through the system until it comes into contact with the working and counter electrodes.

[0132] The devices are preferably arranged in parallel, i.e. the inlet of the system is directly connected to each platinum electrode of each device. The solution to be analysed, introduced into the inlet of the system, enters each device simultaneously and travels through the system until it comes into contact with the working and counter electrodes.

[0133] A detection system allows several measurements to be carried out simultaneously. For example, each detector can be configured to detect the same nucleic acid. Upon introduction of the solution to be analyzed, a detection measurement can be carried out or even the nucleic acid concentration can be measured several times. Such a detection system allows measurements to be obtained with high accuracy.

[0134] 10 shows a schematic example of a detection system 200 comprising eight detection devices 42. Each detection device comprises a liquid inlet 47 and a fluid outlet 48. Each inlet 47 can be directly connected to a system inlet in a parallel arrangement. Each detection device herein comprises two sets of "working and counter electrodes" 50, 52. Each electrode is connected to an electrical connection area 46 that allows the electrodes to be electrically connected to an electrical measurement system.

[0135] The detection system may advantageously comprise two devices arranged to detect two different target nucleic acids, in which case the detection system makes it possible to carry out measurements on the different nucleic acids simultaneously.

[0136] In particular, each device of the system may be configured to detect a different target nucleic acid than the other devices are configured to detect, in which case the detection system allows measurements to be carried out simultaneously on as many different nucleic acids as there are devices in the system.

[0137] Methods for detecting target nucleic acids Another object of the invention is a method for detecting a target nucleic acid in a solution to be analyzed, comprising the following steps: Step (E1) of providing a working electrode, the working electrode being a platinum electrode for detecting the previously presented nucleic acid; Step E2 of providing a counter electrode; a step E3 of electrically energizing the working electrode and the counter electrode to maintain a working voltage between the working electrode and the counter electrode; a step E4 of contacting the working electrode and the counter electrode with the solution to be analyzed; A step E5 of determining the intensity of the measured current between the working electrode and the counter electrode; A step E6 of determining the presence of the target nucleic acid in the solution to be analyzed based on the measured intensity, This is the method.

[0138] Steps E1 and E2 of the detection method can be performed by providing an apparatus for detecting the target nucleic acid as described above.

[0139] Step E3 may be performed using an electrical measurement system configured to apply a potential difference between the electrodes, measure this potential difference, and measure the current between the electrodes.

[0140] Typically, the potential difference between the working electrode potential difference VT and the counter electrode potential difference VCE (VT-VCE) can be selected from a potential difference equal to or greater than -250 millivolts and less than or equal to +250 millivolts, preferably greater than or equal to -250 millivolts and less than or equal to -150 millivolts, preferably greater than or equal to -220 millivolts and less than or equal to -180 millivolts, preferably equal to -200 millivolts.

[0141] Step E4 of contacting the working and counter electrodes with the solution to be analyzed can be carried out statically or dynamically.

[0142] In the static mode, a single block and a fixed amount of solution are placed in contact with the electrodes such that the electrodes are in electrical contact with the solution.

[0143] Dynamically, a continuous flow of the solution to be analyzed is made to flow in contact with the electrodes, such that the electrodes are in electrical contact with the solution flow. This dynamic implementation can be particularly performed by a device for detecting nucleic acids when it comprises a microfluidic channel. The flow of the solution to be analyzed can pass through the channel and come into contact with the electrodes. Typically, the flow of the solution can be generated in the microfluidic channel at a selected value equal to or greater than 0.1 μL / s and equal to or less than 1 μL / s, preferably equal to 0.5 μL / s.

[0144] In all cases, contact with the solution to be analysed is adapted to produce electrical contact between the electrodes with the solution to be analysed.

[0145] The solution to be analysed can be an aqueous solution, in particular a physiological solution.

[0146] As previously presented in connection with FIG. 4, during step E4, if the solution to be analyzed contains a target nucleic acid, a hybridization reaction between the target nucleic acid and the probe nucleic acid takes place on the working electrode.

[0147] The electrochemical properties of the electrode are altered by this hybridization, making it possible to detect the presence of target nucleic acid 36 in the solution being analyzed.

[0148] Step E5 occurs when the working and counter electrodes are brought into contact with the solution to be analyzed such that electrical contact between the electrodes occurs in the solution to be analyzed.

[0149] Step E5 of determining the measured current intensity between the working and counter electrodes can be carried out using an electrical measurement system.

[0150] Step E5 can in particular be carried out by chronoamperometric measurement. FIG. 8 shows an example of a chronoamperometric measurement.

[0151] The graph shown in FIG. 8 was obtained experimentally with different reference solutions by setting the potential difference between the working electrode potential VT and the counter electrode potential VCE (VT-VCE) to -200 millivolts and measuring the electric field strength between these electrodes over time.

[0152] The electric field strength between the electrodes is plotted against the vertical axis, with the horizontal axis corresponding to time.

[0153] Curve 84 represents the case where the solution being analyzed is a reference solution that does not contain any nucleic acids.

[0154] The measurements of curve 84 were carried out before the others.

[0155] The curves 82, 80, 78, 76, 74, 72 and 70 are the same as those shown in Fig. 1. -18 , 10 -16 , 10 -14 , 10 -12 , 10-10 , 10 -8 and 10 -6 This represents the case where the target nucleic acid has a concentration equal to moles / liter.

[0156] After each electrochemical measurement, the electrodes are rinsed with a physiological solution, i.e. a neutral reference solution. It should be noted that the measurements are performed in order of increasing nucleic acid concentration of the reference solutions.

[0157] FIG. 8 shows that different concentrations of target nucleic acid can be distinguished by the average value of the measured intensity over time, the higher the average value, the higher the concentration.

[0158] Step E6 of determining the presence of the target nucleic acid in the analyzed solution can be carried out by comparing the measured intensity to a reference intensity or probe intensity previously measured for a reference solution that does not contain the target nucleic acid. If the measured intensity is significantly different from the probe intensity, the target nucleic acid is present in the analyzed solution. A significant difference is a difference that exceeds a certain threshold. This threshold can be evaluated from this type of statistical measure, allowing the evaluation of the standard deviation and the background noise. The threshold can be selected, for example, equal to one standard deviation or several standard deviations, for example three standard deviations.

[0159] Optionally, the method further comprises the following sub-steps: a first sub-step SE1 of providing two reference solutions having different concentrations of the target nucleic acid; a second sub-step SE2 of measuring a reference intensity between the working electrode and the counter electrode for each reference solution, the electrodes being in contact with the reference solutions and the electrodes being energized to maintain a working voltage between the working electrode and the counter electrode; a third substep SE3 of determining the correspondence of the current intensity between the working electrode and the counter electrode as a function of the target nucleic acid concentration of the solution to be analyzed; The method further comprises the steps of: The method comprises determining the target nucleic acid concentration in the solution being analyzed from the measured intensities using the correspondence relationship.

[0160] During the first substep SE1, two reference solutions with different known concentrations of the target nucleic acid are provided. Preferably, a number of reference solutions is provided that represents more than 2, the higher the number, the better the calibration.

[0161] For example, the reference solution is 10 -18 , 10 -16 , 10 -14 , 10 -12 , 10 -10 , 10 -8 and 10 -6 It may have a target nucleic acid concentration equal to moles / liter.

[0162] During the second sub-step SE2, each reference solution is successively used to contact the working and counter electrodes so as to create electrical contact between the working and counter electrodes.

[0163] A voltage is applied to the working and counter electrodes to maintain a working voltage between them of, for example, -200 millivolts.

[0164] Regarding step E5 of the method for detecting a target nucleic acid, the intensity between the electrodes is measured, which is called the reference intensity since it is determined for one of the reference solutions with known nucleic acid concentration.

[0165] The determination notably comprises the calculation of the average intensity obtained over time by chronoamperometry.

[0166] FIG. 8, discussed above, corresponds to the determination of eight reference intensities, each associated with a known nucleic acid concentration.

[0167] During a third substep SE3, the correspondence between the current intensity and the nucleic acid concentration is determined.

[0168] This correspondence allows one to relate current intensity to nucleic acid concentration, and conversely, nucleic acid concentration to current intensity, at least over a particular range of intensities and a particular range of concentrations.

[0169] This correspondence can be determined, for example, by performing a linear regression of the standard intensities as a function of the nucleic acid concentration.

[0170] FIG. 9 shows another possible correspondence in which the relative intensities are determined before performing the linear regression.

[0171] This relative intensity is composed of the probe intensity defined as the reference intensity for a reference solution containing no nucleic acid.

[0172] Relative intensity is defined as the absolute value of the ratio of the difference between the reference intensity and the probe intensity.

[0173] The experimental values ​​from the experiment associated with FIG. 8 are used to plot the points in FIG.

[0174] A linear regression of these points is performed to obtain the fitting curve 90.

[0175] A fitting curve modeling the relative intensity as a function of nucleic acid concentration constitutes the desired correspondence.

[0176] When a correspondence between current intensity and nucleic acid concentration is obtained, the calibration process is complete.

[0177] Nucleic acid detection methods can be improved to provide estimates of nucleic acid concentration through correspondence.

[0178] The intensity measured for the analyzed solution with unknown nucleic acid concentration constitutes an entry value of a correspondence relationship which makes it possible to relate this entry value to an exit value which is the nucleic acid concentration, this concentration constituting an estimate of the nucleic acid concentration in the analyzed solution.

[0179] The above-mentioned detection method can be carried out in particular when the target nucleic acid is a nucleic acid fragment of a pathogen, for example an RNA nucleic acid fragment encoding a coronavirus. Such a method may be useful in the field of rapid diagnostics in biology during emergencies. The described method makes it possible to detect these fragments at trace levels, which is not possible using electrochemical techniques based on the use of gold electrodes.

[0180] Moreover, in the described method it is possible to obtain quantitative and absolute measurements without using the PCR technique - the polymerase chain reaction - which comprises reverse transcription of RNA to DNA - RT - and amplification of the DNA strand, bringing RNA fragments to a detectable threshold.

Claims

1. A method for attaching nucleic acids to platinum electrodes (5, 20), comprising the following steps: Step (S1) is a step of attaching an ethylenediamine molecule (22) to the electrodes (5, 20), wherein step (S1) includes electrolytic oxidation of the primary amine of the ethylenediamine molecule (22) by cyclic voltammetry. Step (S2) involves attaching a sulfoSMCC molecule (26) of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester to the ethylenediamine molecule (22). Step (S3) of attaching nucleic acid (32) to the sulfoSMCC molecule (26), wherein the nucleic acid (32) is pre-modified to contain a thiol functional group, and during steps (S1), (S2), and (S3), the electrodes (5, 20) come into contact with an aqueous solution, that is, a solution in which the solvent is water, step (S3), A method that includes the following:

2. The method according to claim 1, wherein the aqueous solution is a physiological solution.

3. The method according to claim 1 or 2, wherein the electrodes (5, 20) are microelectrodes at least partially disposed in a microfluidic channel.

4. The method according to claim 1 or 2, further comprising a stabilization step (S4) in which the electrodes (5, 20) are placed in a physiological solution for 20 to 40 minutes after step (S3), wherein the solution has a NaCl concentration of 0.4 mol to 0.6 mol.

5. A platinum electrode (20) for detecting a target nucleic acid (36), A platinum electrode (20) comprising an ethylenediamine molecule (22) attached to the electrodes (5, 20), wherein a sulfoSMCC molecule (26) of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester is attached to the ethylenediamine molecule (22) by electrolytic oxidation of the primary amine of the ethylenediamine molecule (22) by cyclic voltammetry, a probe nucleic acid (32) is attached to the sulfoSMCC molecule (26), the probe nucleic acid (32) contains a thiol functional group, and the probe nucleic acid (32) is complementary to the target nucleic acid (36).

6. A device (100) for detecting a target nucleic acid, comprising a platinum electrode (5), a counter electrode (6), and an electrical measurement system electrically connected to the platinum electrode (5) and the counter electrode (6) as described in claim 5.

7. The apparatus according to claim 6, wherein the apparatus further comprises a microfluidic channel (4), the platinum electrode (5) is a microelectrode, and the platinum electrode (5) and the counter electrode (6) are at least partially located in the microfluidic channel (4).

8. A system (200) for detecting a target nucleic acid, comprising a plurality of devices (100) according to claim 6 or 7, wherein the system (200) includes an inlet configured to receive a solution to be analyzed, and the inlet is connected to a platinum electrode of each device.

9. The detection system (200) according to claim 8, wherein two of the plurality of devices are configured to detect two different target nucleic acids.

10. A method for detecting a target nucleic acid in a solution to be analyzed, wherein the method comprises the following steps: Step (E1) of providing a working electrode, wherein the working electrode is the platinum electrode described in claim 5. Step (E2) to provide a counter electrode, A step (E3) of electrically applying voltage to the working electrode and the counter electrode to maintain the working voltage between the working electrode and the counter electrode, Step (E4) of bringing the working electrode and the counter electrode into contact with the solution to be analyzed. Step (E5) to determine the measured current intensity between the working electrode and the counter electrode. A step (E6) to determine the presence of a target nucleic acid in the solution to be analyzed based on the measured intensity, A method that includes the following:

11. The following sub-processes, A first sub-step (SE1) supplies two reference solutions having different concentrations of target nucleic acids. A second sub-step (SE2) for each reference solution, wherein the reference intensity between the working electrode and the counter electrode is measured, the second sub-step (SE2) wherein the electrode is in contact with the reference solution and a voltage is applied to the electrode to maintain the working voltage between the working electrode and the counter electrode, A third sub-step (SE3) is to determine the correspondence between the current intensity between the working electrode and the counter electrode as a function of the target nucleic acid concentration of the solution to be analyzed. It further includes a corrective process which includes, The method according to claim 10, further comprising the step of determining the concentration of a target nucleic acid in the solution to be analyzed from the measured intensity using the correspondence.

12. The method according to claim 10 or 11, wherein the target nucleic acid is a nucleic acid fragment of a pathogen.

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