WO2024213852A1 - Electrochemical device for enzymatic synthesis of polynucleotides - Google Patents

Abstract

The present invention relates to an electrochemical device (1) for enzymatic synthesis of polynucleotides, comprising at least one circulation duct (4) configured to receive an electrolytic solution, the circulation duct (4) being delimited by a first wall (6) and a second wall (7) opposite each other, the first wall (6) forming a support plate (8) of at least one reaction site configured for the enzymatic synthesis and able to carry a polynucleotide initiator fragment, the circulation duct (4) being equipped with a set of electrodes (2), the set of electrodes comprising at least one electrode of an array of electrodes (10) formed on the support plate (8) and operatively associated with the reaction sites, the set of electrodes (2) also comprising at least one additional electrode (16) arranged on the second wall (7) opposite the support plate (8).

Description

Title of the invention: Electrochemical device for enzymatic synthesis of polynucleotides

Technical field

The present invention relates to the field of electrochemistry, and the application of electrochemistry to a step of a process for the synthesis of oligonucleotides, in particular strands of DNA and/or RNA.

State of the art

DNA and RNA oligonucleotides are short molecules of DNA and RNA, also called polynucleotides. They are linear polymers of nucleotide monomers or analogues thereof that are capable of specifically binding to other oligonucleotides through a regular pattern of monomer interactions. Polynucleotides typically range in size from a few monomers, e.g. 5 to 40 monomers when they are usually called "oligonucleotides", to several thousand monomers. Typically, polynucleotides comprise the four naturally occurring nucleotides, e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose equivalents for RNA, linked by phosphodiester bonds; However, they may also include non-natural nucleotide analogues, for example modified bases, sugars or internucleoside linkages.

The synthesis of these polynucleotide fragments can in particular be obtained by an enzymatic synthesis operation allowing the synthesis of sequences of several hundred nucleotides. Different applications are permitted due to the possibility of obtaining, more quickly and more reliably, longer sequences, including genetic data storage.

Methods for the enzymatic synthesis of polynucleotides are known in which elongation steps and deprotection steps are carried out cyclically at identified reaction sites.

Each elongation step consists of the addition of a nucleotide to the deprotected end of a fragment of a polynucleotide, or to an initiator fragment in the case of the first elongation step, the polynucleotide in a reaction site being obtained by successive additions of the desired nucleotides, nucleotide by nucleotide. Each deprotection step is implemented following an elongation step and aims to deprotect certain polynucleotide fragments, such that the polynucleotide fragments thus targeted are in a state suitable for receiving a new nucleotide during the following elongation step. It is understood that during a deprotection step, it is appropriate to deprotect only the polynucleotide fragments intended to receive a nucleotide during the following elongation step so that localized deprotection steps are carried out.

Localized electrochemical deprotection can be accomplished in a variety of ways. For example, the pH can be controlled locally, for example at a reaction site to deprotect bonds sensitive to these pH values. It is also possible to control the electric potential or electric current between a working electrode present locally at a reaction site and a reference electrode, the deprotection being accomplished by reduction or oxidation of a redox-sensitive bond.

In the case of enzymatic synthesis carried out on support plates comprising a plurality of reaction sites, the electrochemical deprotection is implemented locally using a set of electrodes, respectively associated with one of the reaction sites and among which some are electrically supplied with a potential allowing the redox reaction when an electrochemical deprotection must be implemented in the corresponding reaction site.

The electrode assembly may in particular consist of an assembly known as a two-electrode assembly, with a working electrode and a counter electrode, also forming the reference electrode for determining the value of the electrical potential supplied to the working electrode. A potential is applied only to the working electrode or to the working electrodes which are arranged in reaction sites in which it is desired to generate electrochemical deprotection, no potential being applied to the counter electrode. The potential between the working electrode and the counter electrode is thus controlled without a potential being applied to the latter, the counter electrode being there only to compensate for the electrical stimulus which is applied to the working electrode and to carry out the reverse reaction.

The electrode assembly may also consist of an assembly known as a three-electrode assembly, with a working electrode, a counter electrode, and a reference electrode separate from the counter electrode. The working electrode remains the electrode to which a desired electrical potential is applied, and the potential applied directly between the working electrode and the reference electrode, which is always at a static potential, is controlled. Again, the counter electrode is only there to automatically do the reverse reaction of the working electrode.

In each of these sets, the current flowing between the working electrode and the counter electrode is controlled, and the electrical potential of the working electrode is simultaneously controlled, i.e. the potential difference between the working electrode and the reference electrode.

It is known to arrange enzymatic synthesis devices with reaction sites formed in wells. In this context, the reference electrode, or counter electrode, is arranged around each of the wells, with a working electrode that is arranged at the bottom or in the side wall of each well. Alternatively, the reference electrode can surround the entire wells and the working electrodes housed therein.

In some application cases, a problem with field lines between the working electrodes and the reference electrode has been observed, particularly in the case of a three-electrode system with counter-electrodes each surrounding their own working electrode, particularly when the three-electrode assembly is submerged in an electrolytic solution. The convergence of the field lines from each working electrode to the reference electrode, arranged substantially in the same plane as the working electrodes, increases the resistance and limits the possibility of uniform control of each working electrode.

Furthermore, the integration of the reference electrode, or reference electrodes, in addition to the integration of the counter electrodes, limits the space available on the support plate.

Summary of the invention

The present invention aims to overcome the drawbacks of prior systems, by proposing a device allowing an efficient electrochemical reaction and in a reduced footprint to carry out a step of deprotection of polynucleotide fragments, in particular RNA or DNA, in particular to prepare these fragments for a subsequent elongation step within the framework of an enzymatic synthesis process.

The invention falls within the above context by proposing an electrochemical device for enzymatic synthesis of polynucleotides, comprising at least one circulation conduit configured to receive an electrolytic solution, the circulation conduit being delimited by a first wall and a second wall distinct from each other, the first wall forming a support plate for at least one reaction site configured for enzymatic synthesis and capable of carrying an initiator fragment of polynucleotide, in particular RNA or DNA, the circulation conduit being equipped with a set of electrodes controlled by a control module, the set of electrodes comprising at least one electrode of a network of electrodes formed on the support plate and operationally associated with the reaction sites, the set of electrodes comprising at least one additional electrode arranged on the second wall at a distance from the support plate.

Such an electrochemical device is notably used for a step of deprotection of polynucleotide fragments, notably DNA and/or RNA, which have previously been synthesized on reaction sites arranged in the circulation conduit of the electrochemical device, whatever the shape of these and the means implemented to carry out this synthesis.

The electrochemical deprotection step consists of removing the protection present at the free end of the polynucleotide fragments, in particular DNA and/or RNA, which have just been extended by one nucleotide, by an oxidation-reduction reaction generated by the targeted electrical supply which is made in the direction of one of the electrodes of the electrode network and by the presence of an electrolytic solution in the circulation path on either side of which the electrodes of the set of electrodes are arranged.

The electrolytic solution circulates in the circulation conduit delimited at least by the two separate walls on which the electrodes of the electrode assembly are arranged.

The space on the support plate for the working electrodes, to which the electrical potentials are applied, is thus optimized since the additional electrode(s) are relocated to the wall of the circulation duct opposite the support plate.

The at least one additional electrode which is arranged according to the invention on a wall at a distance from that of the electrode network must be considered as an electrode distinct from the electrode network. The electrodes of the electrode array, just like the additional electrode, may have dimensions that vary from one another, in particular depending on their function and their position. Depending on the size of the electrochemical device, these electrodes may be of the order of a centimeter, a millimeter, a micrometer or a nanometer. As a non-limiting example, the at least one additional electrode may have a dimension that is different from that of the electrodes of the electrode array, with the at least one additional electrode being of the order of a millimeter and the electrodes of the electrode array being of the order of a micron. In the following, we will generally refer to an electrode array without any particular distinction as to size, knowing that such a term may cover an array of microelectrodes.

A positive surface differential between the additional electrode and the working electrodes of the electrode array allows for a reduced internal resistance of the cell, providing a greater degree of process control. Preferably, the surface area of the additional electrode is at least 10 times greater than the combined surface area of the working electrodes included in the electrode array, particularly preferably at least 25, 50, 100 or 1000 times greater. If there are multiple additional electrodes, it is the combined surface area of the additional electrodes that is to be taken into account.

According to an optional feature of the invention, said at least one circulation conduit consists of one conduit or two conduits.

According to a characteristic of the invention, the first wall and the second wall are opposing walls, and the additional electrode is arranged on the second wall opposite the support plate.

Arranging the electrodes on opposing walls, that is to say on either side of the conduit in which the electrolytic solution circulates, makes it possible to have field lines which are all parallel, across the flow of circulation of the electrolytic solution, between the working electrodes and the additional electrode arranged on the opposite wall of the circulation conduit and a stimulus distributed uniformly on all the working electrodes.

According to an optional feature of the invention, the first wall (6) and the second wall (7) are spaced apart by a distance of 10 μm to 1 cm. Positioning the two walls at such a distance provides optimal conditions for an enzymatic elongation reaction of a polynucleotide fragment, by allowing efficient deprotection while limiting the degradation of the reagents such as enzymatic precipitation. Thus, it it is possible to carry out the elongation reaction and the deprotection reaction in the same reaction site within the circulation conduit. In particular, the first wall (6) and the second wall (7) may be spaced apart by a distance of 10 pm to 200 pm, more particularly 10 pm to 100 pm or 10 pm to 50 pm. The first wall (6) and the second wall (7) may also be spaced apart by a distance of 100 pm to 1 cm, more particularly 200 pm to 1 cm or 500 pm to 1 cm.

In a particular embodiment, the device is configured such that the distance between the first wall (6) and the second wall (7) is adjustable. For example, the first wall (6) and/or the second wall (7) may be movable so as to be able to modify the distance separating them, to increase or decrease this distance depending on the use of the device. Thus, said distance may be modified depending on the type of reaction or the reaction step carried out using the electrochemical device. According to an optional characteristic of the invention, the first wall carries several reaction sites, each reaction site being operationally associated with at least one electrode of the electrode network.

According to an optional characteristic of the invention, the circulation conduit is further delimited by a side wall connected at least to one or the other of the first wall and the second wall, the side wall carrying at least one other additional electrode.

According to an optional feature of the invention, the support plate is configured to support at least one semiconductor, the electrode array being arranged on said semiconductor.

The support plate may in particular comprise a printed circuit board which carries components which may in particular take the form of CMOS (Complementary metal-oxide-semiconductor) or Screen-Printed Electrode (SPE) type semiconductors. Alternatively, the support plate may consist of a glass plate.

According to an optional feature of the invention, the electrode network is configured so as to comprise at least one working electrode operationally associated with a reaction site and controlled by a control module to form an anode or a cathode capable of carrying out an oxidation-reduction operation.

This control is carried out remotely using a potentiostat or an electric generator. In the particular case of a CMOS, the assembly is the same except for adding a logic gate system after the generator so that you can apply the current or potential values you want (and where you want). Depending on the values distributed by the generator coupled to the architecture of the transistors making up the CMOS, the value of the currents or potentials at the electrodes placed at the output, which are in contact with the solution, can be negative, forming a cathode. If this value is positive, an anode is formed.

According to an optional feature of the invention, the electrode network is configured so as to comprise several working electrodes which are respectively operationally associated with a reaction site and controlled by a control module to form an anode or a cathode capable of carrying out an oxidation-reduction operation, each of the working electrodes being able to be controlled independently of the others.

By way of example, without this being limiting of the invention, a first working electrode can be driven to form an anode to which a positive potential of IV is applied, while a second working electrode can be driven to form a cathode to which a negative potential of 2V is applied and a third working electrode can be driven to form an anode to which a positive potential of 2V is applied.

According to an optional feature of the invention, the electrode network and the at least one additional electrode are electrically linked.

According to an optional characteristic of the invention, the electrode network and the at least one additional electrode are independent.

According to an optional characteristic of the invention, the set of electrodes comprises two distinct types of electrodes including one or more working electrodes and one or more counter-electrodes associated with the working electrodes, the at least one additional electrode being a counter-electrode type electrode.

In such a two-electrode arrangement, it can be considered that the reference electrode is formed by the counter electrode, the cables connecting the reference electrode are fused with the counter electrode cables.

The control, that is to say the application of the desired values of current or potential, is applied directly to one or more working electrodes, and the counter electrode automatically finds itself at a potential of opposite value. The counter-electrode is defined as electrodes in the electrode set as electrodes to which no potential is applied directly. Once it is chosen to apply a given potential to an electrode, it must change its name regardless of its previous role and must be considered as a new working electrode.

According to an optional characteristic of the invention, the set of electrodes comprises three distinct types of electrodes including one or more working electrodes, one or more counter-electrodes associated with the working electrodes, and at least one electrode of the reference electrode type.

According to an optional characteristic of the invention, the at least one additional electrode is an electrode of the reference electrode type and/or an electrode of the counter-electrode type.

As mentioned above, the control is carried out on the working electrode and the counter electrode uses the opposite value to compensate/neutralize the electrical system as a whole. The reference electrode will allow the working electrode to be polarized by setting fixed potential values that do not vary during stimulation and relaxation. It serves as a reference point for the potential values used while not chemically and electrically participating in the reducing/oxidizing mechanisms of the solution.

When several additional electrodes are arranged on the second wall, opposite the network of electrodes formed on the support plate, these may all be of the same type, that is to say all reference electrodes or all counter electrodes, or they may be of different types and form a mix of reference electrodes and counter electrodes arranged in a coherent manner with respect to the arrangement of the working electrodes of the network of electrodes formed on the support plate.

According to an optional feature of the invention, the electrode network comprises several electrodes of the working electrode type and of the counter-electrode type, an electrode of the first type being electrically supplied so as to form a cathode or an anode and surrounding at least one electrode of the second type electrically supplied so as to form an anode or a cathode.

A first embodiment may consist of an electrode network that comprises pairs of working electrodes and counter electrodes, forming anode and cathode pairs, with the pairs being independent of each other. In a manner Alternatively, a second embodiment may consist of an arrangement where a counter electrode surrounds all the reference electrodes. In the latter case, we speak of a universal counter electrode which is simpler to integrate into the set of electrodes.

According to an optional characteristic of the invention, the electrochemical device comprises a plurality of electrodes of the working electrode type, the network of electrodes formed on the support plate comprises a plurality of electrodes, the electrochemical device comprising on the second wall a single additional electrode, common to the plurality of electrodes of the working electrode type.

According to an optional characteristic of the invention, the electrochemical device comprises a plurality of electrodes of the working electrode type, the network of electrodes formed on the support plate comprises a plurality of electrodes, the electrochemical device comprising on the second wall a plurality of additional electrodes respectively associated with at least one of the electrodes of the plurality of electrodes of the working electrode type.

According to an optional feature of the invention, the working electrode is supplied so that the supply current has a value greater than a threshold value, such that the working electrode is made active for the production of protons or for the depletion of protons.

When it is desired that a deprotection of the polynucleotide fragment, in particular DNA and/or RNA, present in the reaction site takes place, the power supply of the working electrode associated with this reaction site is controlled so that the current has a value greater than a given corresponding threshold value. The precise value at which to activate the electrochemical system is known, this precise value depending on the electrolytic solution used. For example, a positive potential of a value equal to IV is applied to half of the electrodes, i.e. of the order of +1V relative to the standard hydrogen electrode. This value is sufficient to activate the production of protons thanks to the electrolytic solution used. Protons are thus produced on each of the sites associated with the electrodes where this potential has been applied, i.e. on half of the sites in the example. Polynucleotide fragments, including DNA and/or RNA, placed on the support plate were deprotected at half of the sites. These fragments can be extended in the next enzymatic synthesis cycle. When one wishes to ensure that no deprotection action will be performed on the polynucleotide fragment present in a reaction site, two choices are possible. The first is to do nothing, and therefore to not apply any electrical potential to the electrodes associated with the reaction sites in which the polynucleotide fragment must not be deprotected. The second choice is to apply a precise potential value that is known to be insufficient to activate the production of protons for the electrolytic solution used, or that is known not to produce enough protons to completely deprotect the polynucleotide fragments. In the example mentioned above, one can apply to the other half of the reaction sites a positive potential of a value equal to 0.5V, this value being insufficient to produce enough protons to lower the pH around the sites. The polynucleotide fragments present in this other half of the sites are thus not deprotected and they cannot therefore be extended in the following enzymatic synthesis cycle.

Another approach is to use proton depletion. When we apply a potential higher than a threshold value to certain electrodes, we will have a generation of basic species on each of the sites associated with the electrodes where we applied this potential and therefore an increase in the pH around these sites. There will therefore be a deprotection at the level of these sites.

According to an optional feature of the invention, the at least one additional electrode projects from the second wall of the circulation conduit. In other words, the at least one additional electrode tends to move closer to the support plate forming the first wall, so that it is more exposed to the flow of the electrolytic solution present in the circulation conduit.

According to an optional feature of the invention, the at least one additional electrode is arranged in a recess in the second wall of the circulation conduit.

It is understood that this characteristic covers both a positioning of the additional electrode flush with the second wall and a positioning of the additional electrode in a well dug in the second wall, for example at the level of a bottom wall of this well.

According to an optional feature of the invention, the second wall comprises several additional electrodes and at least one of these additional electrodes projects from the second wall of the circulation duct while at least one of these additional electrodes is arranged in a recess of this second wall. According to an optional characteristic of the invention, the at least one additional electrode is a single electrode extending opposite several electrodes of the electrode network and having a shape chosen from the shapes of a plate, grid, spiral in contact with the second wall of the circulation duct.

According to an optional characteristic of the invention, the at least one additional electrode comprises several additional electrodes extending respectively opposite an electrode of the electrode network and respectively having a shape chosen for example a rectangular or annular shape.

According to an optional feature of the invention, the additional electrode is transparent. Alternatively, the additional electrode may be opaque or may present a compromise between opacity and transparency.

According to an optional feature of the invention, the electrodes of the electrode network and/or the additional electrode are made from a metal, an oxide or a combination of metal and oxide. As a non-limiting example, these electrodes may be formed from platinum, gold or indium-tin oxide.

According to an optional feature of the invention, the electrodes of the electrode network can be obtained by spraying, electrodeposition or even photo-modeling.

According to an optional feature of the invention, the circulation conduit comprises at least one semi-permeable membrane. This semi-permeable membrane is intended to isolate the species generated at the electrodes of the wall. These membranes can be glass membranes, ionic polymer membranes. They can be transparent so as not to disturb any attempt at optical characterization.

Additionally, it is possible to plan to add an ion exchange membrane within the circulation conduit, between the first wall and the second wall, in addition to the semi-permeable membrane.

Alternatively, without departing from the context of the invention, the circulation conduit may be empty and only dedicated to the circulation of fluid between the two opposing walls on which the electrodes are arranged. The invention also relates to a method for synthesizing polynucleotides by the electrochemical device for enzymatic synthesis as previously mentioned, comprising an association step, during which each reaction site of a support plate is operationally associated with an electrode of the electrode network, and a deprotection step during which a labile protective group of a polynucleotide fragment, in particular RNA or DNA, previously formed on a reaction site is removed by an oxidation-reduction operation carried out in contact with an electrode of the electrode network, the deprotection step comprising an application of an appropriate potential to at least one electrode of the electrode network.

The polynucleotide fragment subject to a deprotection step may consist of an initiator fragment or a set formed of an initiator fragment and a series of one or more triphosphate nucleotides respectively added during an elongation step. The elongation step may be carried out cyclically, alternating with a deprotection step, it being understood that the nucleotide added during the elongation step carries a labile protective group, which should be removed if a new elongation operation is desired.

According to an optional characteristic of the invention, the deprotection step and an elongation step, during which a nucleotide is added to the polynucleotide fragment, in particular RNA or DNA, from which the labile protective group was removed during the deprotection step, are repeated until a polynucleotide of interest is obtained.

According to an optional feature of the invention, the labile protecting group is sensitive to a change in pH, the application of the potential difference of the deprotection step resulting in a change in pH leading to the cleavage of the labile protecting group.

Drawings

Other characteristics and advantages of the invention will become apparent from the description which follows on the one hand, and from several examples of embodiment given for information purposes and without limitation with reference to the attached schematic drawings on the other hand, in which:

[fig. 1] is a flowchart illustrating certain steps of an enzymatic synthesis process, including a step of elongation of a polynucleotide fragment, in particular RNA or DNA, and a deprotection step using an electrochemical device according to the invention;

[fig. 2] is a schematic representation of an electrochemical device according to the invention, illustrating a conduit for circulating electrolytic solution with a network of electrodes arranged on a first wall delimiting this conduit and at least one additional electrode arranged on a second wall opposite the first wall;

[fig. 3] is a schematic representation of electrodes of the electrode network arranged on the first wall delimiting the conduit of the electrochemical device according to the invention;

[fig. 4] is a schematic representation, exploded, of an exemplary embodiment of an electrochemical device according to the invention;

[fig. 5] schematically illustrates an advantage of the electrochemical device according to the invention, with parallel field lines extending across the circulation conduit;

[fig. 6] schematically illustrates a circulation conduit of an electrochemical device according to one embodiment, with a first wall delimiting the circulation conduit which comprises working electrodes surrounded respectively by a counter-electrode, these two types of electrodes having an annular shape, and with a second opposite wall which comprises reference electrodes;

[fig 7] schematically illustrates a circulation conduit of an electrochemical device according to other embodiments, with a first wall delimiting the circulation conduit which comprises sub-assemblies formed by two concentric working electrodes, and with a second opposite wall which comprises reference counter-electrodes (7 A), or a second opposite wall which comprises reference electrodes (7B) or counter-electrodes (7C);

[fig 8] schematically illustrates a circulation conduit in which different arrangements of electrodes and their electrical supply are represented to illustrate the potential differences which can be applied on either side of the circulation conduit to generate the electrochemical reactions necessary for a deprotection step of an enzymatic synthesis;

[fig 9] schematically illustrates a second wall of an electrochemical device according to the invention on which several possible arrangements of electrodes are represented. additional, with either counter electrodes alone, or reference electrodes alone, or combinations of counter electrodes and reference electrodes;

[fig. 10] schematically illustrates a first wall of an electrochemical device according to the invention; several possible arrangements of an electrode network are shown, with working electrodes which are either alone or in combination with counter-electrodes or reference electrodes;

[fig. 11] schematically illustrates a second wall of an electrochemical device according to the invention on which an additional electrode in the form of a grid is represented;

[fig. 12] schematically illustrates a second wall of an electrochemical device according to the invention on which an additional electrode in the form of a spiral is represented;

[fig. 13] schematically illustrates a second wall of an electrochemical device according to the invention on which several types of arrangements of additional electrodes are represented by way of example;

[fig. 14] schematically illustrates a wall of an electrochemical device according to the invention on which two types of electrodes of an electrode network are represented, a first type of electrodes in annular form and a second type of electrode in grid form,

[fig. 15] schematically illustrates two circulation conduits of an electrochemical device according to an embodiment with a semi-permeable membrane, with a first wall delimiting the circulation conduit which comprises working electrodes, these electrodes having an annular shape, and with a second opposite wall which comprises reference electrodes for the first conduit and counter electrodes for the second conduit,

[fig. 16] schematically illustrates a prior art system using two concentric annular platinum electrodes, the external electrode being the reference counter electrode and the internal electrode the working electrode,

[fig 17] schematically illustrates another prior art system using the same electrodes as the system of figure 16 but with the role of the electrodes reversed; thus, the reference counter electrode is the internal electrode and the working electrode is the external electrode,

[fig. 18] illustrates a third prior art system using the same configuration as the system of figure 17 with in addition a platinum reference electrode at a distance and upstream of the working electrodes, thus in this system the counter electrode does not act as a reference electrode,

[fig. 19] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 16,

[fig. 20] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 17,

[fig. 21] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 19,

[fig. 22] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 7 where the additional electrode is made of ITO,

[fig. 23] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 6 where the additional electrode is made of ITO,

[fig. 24] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 7 where the additional electrode is gold,

[fig. 25] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 6 where the additional electrode is gold,

[fig. 26] shows a cyclic voltammetry curve recorded at a working electrode of the system of figure 7 where the additional electrode is made of platinum, it turns out that this curve is identical to that recorded at a working electrode of the system of figure 6 where the additional electrode is made of platinum, which is why only one curve is shown here in order to avoid redundancies.

It should first be noted that while the figures set out the invention in detail for its implementation, these figures can of course be used to better define the invention, where appropriate. It should also be noted that these figures only set out examples of embodiments of the invention. Detailed description

The features, variants and different embodiments of the invention may be combined with each other, in various combinations, to the extent that they are not incompatible or mutually exclusive. In particular, variants of the invention may be imagined comprising only a selection of features described below in isolation from the other features described, if this selection of features is sufficient to confer a technical advantage or to differentiate the invention from the state of the art.

In the figures, elements common to several figures retain the same reference.

As a reminder, the invention aims to protect an electrochemical device comprising a set of electrodes such that electrodes are arranged on either side of a conduit for circulating an electrolytic solution, i.e. on walls arranged in opposition to delimit the conduit. This electrochemical device is used in the context of an enzymatic synthesis of polynucleotides and more particularly for an electrochemical deprotection step which is part of an enzymatic synthesis process as illustrated schematically in FIG. 1.

A cycle of the enzymatic synthesis method 100, leading to the addition of a nucleotide to a polynucleotide fragment, comprises two successive steps, corresponding respectively to an elongation step 101 and to a deprotection step 102. In summary, during the elongation step, a nucleotide comprising a protective group is added to an initiator or to a polynucleotide fragment already in the process of being formed. Then, the protective group can be removed from this newly added nucleotide, during the deprotection step, in order to be able to carry out additional cycles on this same polynucleotide fragment.

In the method, initiator moieties are provided attached to a solid support. These initiator moieties, which have deprotected free 3'-hydroxyl groups, are respectively arranged in one of the reaction sites formed on the solid support, or support plate. In other words, each reaction site formed on the support plate is capable of carrying a polynucleotide initiator moiety. As used herein, an "initiator moiety" generally refers to a short oligonucleotide sequence with a free 3' end, which can be further elongated by a template-free polymerase, such as a TdT polymerase. In one embodiment, the Initiator fragment is a DNA initiator fragment. In another embodiment, the initiator fragment is an RNA initiator fragment. In some embodiments, an initiator fragment has between 3 and 100 nucleotides, particularly between 3 and 20 nucleotides. In some embodiments, the initiator fragment is single-stranded. In other embodiments, the initiator fragment is double-stranded. In some embodiments, an initiator fragment may comprise a non-nucleic acid compound having a free hydroxyl to which a TdT polymerase can couple a 3'-O protected dNTP nucleotide, e.g., Baiga, U.S. Patent Publications US2019/0078065 and US2019/0078126.

Prior to the elongation step 101, polynucleotides to be treated 103 are considered, whether they are the initiator fragments or the initiator fragments elongated in a previous cycle, and it is determined whether a nucleotide, and what type of nucleotide, must be added to the polynucleotide to be treated.

More particularly, during the elongation step 101, a 3'-O protected dNTP nucleotide and a template-free polymerase, such as a TdT polymerase or a variant thereof, are added to the polynucleotide to be treated under conditions effective for the enzymatic incorporation of the protected nucleotide onto the 3' end of the previously discussed initiator fragments. This reaction produces in the targeted reaction sites elongated initiator fragments, the 3'-hydroxyls of which are protected, 104.

A verification step 106 is then carried out to determine whether the polynucleotide fragments, in particular RNA or DNA, present in each of the reaction sites are complete, i.e. whether they respect the desired length. If in a reaction site, the elongated initiator fragment contains a complete sequence, then the desired sequence can be cleaved from the original initiator fragment. Such a cleavage operation 108 can be carried out using any of a variety of cleavage techniques, for example by inserting a cleavable nucleotide at a predetermined location in the original initiator fragment.

If in a reaction site, the elongated initiator fragment does not contain a complete sequence, then the protection group of the last added nucleotide is removed during a deprotection step 102. The deprotected elongated initiator fragments, with a free 3'-hydroxyl, form polynucleotides to be treated 103 intended to undergo at least one further elongation step 101. Protecting groups are electrochemically labile groups. In other words, deprotection is accomplished by changing the electrochemical conditions in the vicinity of the protecting group, which results in the separation of these groups, just as can be the case in the cleavage operation. An example of such protecting groups is an amino group, i.e. deprotection converts an —O—NH2 group to —OH.

In the invention, these changes in electrochemical conditions are caused by changing or applying an electrical potential that has the effect of, for example, generating an increase or decrease in the pH of an electrolytic solution. In some embodiments, the electrochemically labile groups comprise, for example, pH-sensitive protecting groups that are cleaved whenever the pH is changed to a predetermined value. In other embodiments, the electrochemically labile groups comprise protecting groups that are cleaved directly whenever the reducing or oxidizing conditions are changed, for example, by increasing or decreasing a voltage difference at the site of the protecting group.

The pH change of the electrolytic solution may occur via a redox reaction involving one or more electrochemically active molecules present in the electrolytic solution. For example, the electrolytic solution may include one or more redox species such as quinones (See Thomas Finley, “Quinones,” Kirk-Othmer Encyclopedia of Chemical Technology, 1-35 (2005)). The benzoquinone/hydroxyquinone pair and their derivatives are notably used to produce proton gradients at the surface of an electrode

It should be understood that the enzymatic synthesis process has been described briefly and is not limited to these elongation, deprotection and cleavage steps, although this is not essential for understanding the description that follows. Thus, the enzymatic synthesis process may also comprise one or more capping steps as well as washing steps after the elongation step and/or after the deprotection step.

The electrochemical device 1 according to the invention notably allows the implementation of the deprotection step 102. As illustrated in Figure 2, the electrochemical device 1 according to the invention comprises a set of electrodes 2 configured such that electrodes are arranged on either side of a circulation conduit 4 configured in particular for the circulation of an electrolytic solution according to arrow F.

The circulation conduit 4 is delimited by different walls so as to generate a closed passage section suitable for the circulation of the electrolytic solution, these different walls comprising in particular a first wall 6 and a second wall 7 arranged in opposition, and visible in figure 2.

The first wall 6 is partly formed by a support plate 8 on which are fixed initiator fragments, or polynucleotides to be treated 103 as mentioned above, which are extended cycle after cycle by the addition of successive nucleotides, this addition being made possible by a prior deprotection operation being implemented efficiently by the electrochemical device 1 of the invention and its particular arrangement of the set of electrodes 2.

The support plate 8 may consist of a glass plate, or a printed circuit board, with at least one semiconductor element on which an array of electrodes 10 or microelectrodes is arranged. The description that follows will be carried out by considering a CMOS chip type semiconductor element technology, but it should be noted that other technologies can be considered without departing from the context of the invention as long as they make it possible to integrate an array of electrodes 10 or microelectrodes, capable of interacting with additional electrodes arranged at a distance as they will be described below. As a non-limiting example, the present invention could be implemented with a so-called SPE system in which platinum rings are directly welded to a network of cables located inside the chip and directly connected to electrical cables linked to a potentiostat. Two concentric platinum rings form an assembly in which a working electrode is formed by one of the rings, for example the ring of smaller diameter, the other ring forming both a counter electrode and a polarized reference electrode.

According to the invention, the electrode network 10 is arranged on the support plate 8 forming part of the first wall 6, and at least one additional electrode 16, which will be described in more detail below, is present on the second wall 7 opposite the first wall 6. More particularly, the electrode array 10 is arranged on the semiconductor element of the support plate, with the possibility of arranging a multitude of electrodes with a reduced pitch between them, to allow large-scale enzymatic synthesis. In this context, the electrodes of the electrode array, as is also the case for the additional electrodes which will be mentioned below, can have dimensions in centimeters, millimeters, or microns. It could also be envisaged that the electrodes of the electrode array, or microelectrode array as it may also be called in this description, have nanometric dimensions.

The electrode array 10 is arranged on the support plate 8 in such a way that the electrodes can be exposed to the electrolytic solution circulating in the circulation conduit 4 and be the basis of an oxidation-reduction reaction for example.

The electrode network 10 comprises at least one working electrode 12 on each of the reaction sites arranged on the support plate 8. In other words, an enzymatic synthesis operation implementing the electrochemical device of the invention begins with an association step during which each reaction site of a support plate is operationally associated with an electrode, and in particular a working electrode, of the electrode network.

A control module 14, shown schematically in FIG. 2 at a distance from the first wall but which could be housed there without departing from the context of the invention, is associated with the electrode network 10 to control the selective supply of the working electrodes 12 associated with each of the reaction sites.

In an embodiment which is illustrated in particular in FIG. 3, two working electrodes 121, 122 are arranged concentrically for each pixel 81, i.e. for each reaction site on the support plate 8. The various MOSFET transistors, p-type and n-type, embedded on the CMOS chip 84 participating in forming this support plate 8, make it possible to assign positive current values to each smaller diameter ring and negative current values to each larger diameter ring, it being understood that the electrochemical deprotection can be implemented according to the invention with inverted values, i.e. with negative current values to each smaller diameter ring and positive current values to each larger diameter ring. It is particularly interesting in this context that the ring forming the external working electrode, i.e. the working electrode which surrounds the other, is the electrode which functions as a cathode. On pixel 81 forming a site reaction, the working electrodes can be associated with sensors 83 allowing the progress of the electrochemical reaction to be measured and controlled.

The current induced by the control module 14 is coupled with electrochemistry, that is to say with the electrolytic solution circulating in the circulation conduit 4, to locally generate protons in order to reduce the pH and generate the cleavage of the ends of the polynucleotide fragments present in the reaction sites.

The additional electrode(s) 16 previously mentioned, which are said to be additional insofar as they are not arranged on the semiconductor forming the first wall 6, may also be, depending on the characteristics of the set of electrodes 2 of the electrochemical device 1, a counter electrode 18 and/or a reference electrode 20.

The additional electrode(s) 16 may be made of an inert material, such as platinum, gold, or indium tin oxide (ITO). Gold has the advantages of being easier and less expensive to manufacture than platinum and has a high conductivity which allows the thickness to be reduced while maintaining the same level of control. In addition, by reducing the thickness, the material becomes semi-transparent which makes optical applications possible. ITO has the advantage of being transparent which is useful when one wishes to combine electrical stimulation/activation or analysis with optical stimulation/activation or analysis.

A counter electrode 18, also called an auxiliary electrode, is an electrode used to close the current circuit in the electrochemical cell, in addition to a working electrode. It is generally made of an inert material, for example platinum (Pt), gold (Au), copper (Cu), nickel (Ni), cobalt (Co), mercury (Hg), indium tin oxide (ITO), fluorine doped indium tin oxide (FTO), graphite or glassy carbon, and it is not specifically supplied with current by the control module 14. The current thus flows between the working electrode 12 and the counter electrode 18, in particular via the electrolytic solution present in the circulation conduit 4, and the total surface area of the counter electrode 18 is generally larger than the surface area of the working electrode 12 so as not to be a limiting factor in the kinetics of the electrochemical process. In a two-electrode electrochemical device, the working electrode 12 and the counter electrode 18 form the two types of electrodes used. As an example, the electrochemical device schematically illustrated in Figure 2 is an electrochemical device with two electrodes and the additional electrode is formed by a counter electrode 18, the electrode network 10 being made up only of working electrodes 12.

In a three-electrode electrochemical device, such as can be schematically shown in FIG. 6, for example, a reference electrode 20 is provided as an electrode with a stable and well-known electrical potential and is used as a reference point in the electrochemical cell for controlling and measuring the potential. In these examples, the counter electrode 18 has a shape equivalent to that of the corresponding working electrode 12, here an annular shape, by having a larger diameter than that of the working electrode 12 and by surrounding the corresponding working electrode.

In particular, it can be expected that the reference electrode is polarized, i.e. its potential varies, or a current flows through it, during the electrochemical reaction. As a result, the connection to the motherboard is not a perfect ground connection. The measured potential values are not absolute and change depending on the size of the electrodes, the conductivity of the solution and/or the concentrations of the species during stimulation.

The control module 14 is configured to compensate for these deficits, for example by distributing to the surface of the counter electrode the inverse current value of a current value imposed or measured at the reference electrodes. For example, the reference electrode and the counter electrode can be constantly polarized at +/- 1.4 V so as to be able to compensate for the currents applied to the working electrodes on the CMOS and maintain its internal potential at the same value throughout the synthesis. In this way, the reference electrode can here adopt the electrical properties of a non-polarized reference electrode, while playing the role of counter electrode on the surface of which species are generated throughout the deprotection. In this case, we speak of a reference counter electrode 18, 20 because it plays both roles.

In connection with the above, regardless of the embodiment, the control module 14 receives information relating to the location of the reaction sites in which it is appropriate, for a given deprotection step, to carry out the deprotection of the polynucleotide fragment being formed in these reaction sites, and the control module 14 consequently generates an instruction for electrical supply of the working electrode(s) 12 arranged in said reaction sites. And as mentioned, a reference counter-electrode can be constantly polarized at a value, here at 1.4V, allowing to compensate the currents applied to the working electrodes and to keep a constant internal potential.

The working electrode 12 is distinguished in the electrode assembly 2 of the electrochemical device 1 as being the electrode on which the reaction of interest occurs. Common working electrodes 12 may be made of different materials including, but not limited to, gold (Au), silver (Ag), platinum (Pt). The working electrode may comprise a coating for fixing molecules and in particular those constituting the initiator fragments previously mentioned.

As mentioned previously, the support plate 8 participates in delimiting a circulation conduit 4 so that the CMOS chip 84 and the electrode network 10 that it carries are arranged on the first wall 6, in contact with an electrolytic solution caused to circulate in the circulation conduit 4.

In addition to the electrode array 10 arranged on the first wall 6, and regardless of the type of semiconductor used, the set of electrodes 2 of the electrochemical device 1 according to the invention therefore comprises at least one additional electrode 16 which is arranged on a second wall 7 which is distinct from the first wall, and which is here more particularly opposite the electrode array. In this way, the electrode array 10 on the one hand and the additional electrode(s) 16 on the other hand are arranged on either side of the circulation conduit 4.

In this first example of an electrochemical device, the additional electrode is electrically connected to the CMOS chip, and the electrical connection of this additional electrode to the motherboard is made via the CMOS chip.

Various arrangements of the electrode array and the additional electrode(s) will now be described. These arrangements will be described in relation to the configuration illustrated in FIG. 5, with two opposing walls including the first wall 6 defined at least in part by the support plate 8 and the second wall 7, and with the working electrodes which are arranged on the first wall 6 and the additional electrode(s) 16, whether a counter electrode and/or a reference electrode, which are arranged on the second opposite wall 7. In this way, field lines 17 extend perpendicular to the electrolytic solution path.

In a first example illustrated in FIG. 6, the electrode network 10 present on the first wall 6 comprises working electrodes 12, here nine in number, being understood that this may not be representative of the total number of electrodes especially in the case of a microelectrode array, and counter electrodes 18 which are respectively arranged around one of the working electrodes 12. In this example, each electrode of the electrode array 10 is annular and a working electrode 12 and a counter electrode 18 form a subset 19 of concentric annular electrodes, with the counter electrode surrounding the working electrode. In this example, the working electrode 12 is electrically powered so as to operate as an anode, and the counter electrode 18 forms a cathode, but it should be noted that this could be the reverse, if one wishes to implement a deprotection which involves reducing species, with the working electrode 12 being electrically powered so as to operate as a cathode and the counter electrode 18 forming an anode.

In this example, the additional electrode(s) 16 are reference electrodes 20 and they form a matrix here, on a wall at a distance from the CMOS chip on which the electrode array 10 is arranged. In accordance with what has been mentioned previously, the reference electrodes are advantageously connected to the motherboard, independently of the electrode array 10, it being understood that a connection to the CMOS chip would be possible.

In a second example illustrated in FIGS. 7A, 7B and 7C, the electrode array 10 present on the first wall 6 differs from what has just been described in that the subassemblies 19, again nine in number by way of example, are formed by pairs of working electrodes, with a first working electrode 121 and a second working electrode 122, concentric. In this example, again, each electrode of the electrode array 10 is annular. In accordance with what has been described with reference to FIG. 3, each subassembly 19, which corresponds to a reaction site, can be configured such that the first working electrode 121 is electrically powered so as to function as an anode, and that the second working electrode 122 is electrically powered so as to function as a cathode.

In a particular example, illustrated in Figure 7B, the additional electrode(s) 16 are reference electrodes 20 and they form here a matrix, on a wall at a distance from the CMOS chip on which the electrode array 10 is arranged. In accordance with what has been mentioned previously, the reference electrodes are advantageously connected to the motherboard, independently of the electrode array 10, it being understood that a connection to the CMOS chip would be possible. In another particular example, illustrated in Figure 7C, the additional electrode(s) 16 function as counter electrodes 18.

The electrodes of the electrode network 10 and the at least one additional electrode 16 can be manufactured by different processes and for example by spraying, electrodeposition, photopatterning, without this list being exhaustive.

Without departing from the context of the invention, the different electrodes arranged on opposite walls of the electrolytic solution circulation conduit may be transparent or opaque.

As just presented, the set of electrodes 2 within the meaning of the invention is configured so that the activation of one or more of the working electrodes present in the electrode network 10, via the control module 14, makes it possible to implement electrochemical reactions, for example to generate local modifications of PH so as to carry out operations of deprotection of free ends of polynucleotide fragments or operations of cleavage of polynucleotide fragments to separate them from their reaction site.

The additional electrode(s) 16 located on the surface opposite the electrode array 10 used for synthesis may be used to modify electric fields, measure pH, and provide pH adjustments that compensate for any changes in the performance or conditions of the microelectrode array. For this purpose, the additional electrode(s) 16 may operate independently or be used in concert with electrodes in the electrode array, or provide a feedback loop.

An arrangement of the set of electrodes 2 according to the invention, with an array of electrodes 10 and at least one additional electrode 16 arranged on either side of the circulation conduit 4, can also make it possible to form two addressable zones on either side of the circulation conduit 4, and this can be used for new applications such as controlling the movement of DNA or RNA for example, for gene assembly, sub-pooling or cleavage of DNA or RNA.

In these configurations, the additional electrodes may be electrically connected to the microelectrode array or may be connected independently of the microelectrode array as illustrated in Figure 4. The configuration of Figure 4, with independent connection, is thus less practical to implement since on the one hand the cable linked to the reference counter electrode present on the second wall 7 must be soldered to the motherboard, and on the other hand a platinum wire must be introduced into the fluidic part without causing leaks. However, these drawbacks can be considered minor compared to the advantages of having one or more reference counter electrode(s) at a distance from the CMOS chip and connected independently, since this allows in particular to have greater freedom at the design level and to place this or these reference counter electrode(s) where desired, either on a wall opposite the CMOS chip, forming a ceiling of the electrolytic solution circulation conduit, or on an adjacent side wall of the first wall carrying the CMOS chip. In addition, such a configuration with one or more additional electrodes arranged at a distance from the working electrodes present on the CMOS chip makes it possible to isolate the species formed on the surface of these additional electrodes by keeping them at a distance deemed sufficient and/or by using a membrane. Finally, this makes it possible to simplify the design of the CMOS chip and therefore its cost, since only the connection of the working electrodes must be taken into account here, and this makes it possible to add more reaction sites on the CMOS chip with this space left free by the offset of the reference counter-electrodes.

Figure 8 illustrates different configurations of the electrode assembly whose power supply is controlled by the control module. This control is carried out remotely using a potentiostat or an electric generator. In the particular case of an electrode array mounted on a CMOS component, a logic gate system is provided between the generator or potentiostat and the CMOS component so as to be able to apply desired current or potential values to the desired reaction sites. If this distributed value is positive, an anode is formed with the working electrode as was mentioned with reference to Figure 6 for example. If this distributed value is negative, the working electrode forms an anode.

The control module can thus generate and transmit instructions, via the CMOS and the logic gates, which are different from one electrode to another. Thus, two or more working electrodes 12 can be polarized differently, with for example a first working electrode whose potential is brought to +1V, a second working electrode whose potential is brought to -IV and a third working electrode whose potential is left zero. Thus, the same working electrode can, depending on the case, be configured to operate as an anode or a cathode. Alternatively, it may be provided that working electrodes have an immutable configuration and can only operate as an anode or a cathode.

The value of the potential applied to a working electrode associated with a reaction site is chosen according to the electrolytic solution so that a deprotection of the polynucleotide fragment, in particular RNA or DNA, present in this reaction site takes place. The power supply of the corresponding working electrode is such that the current has a value greater than a given corresponding threshold value. For example, a positive potential of a value equal to IV, i.e. of the order of +1V relative to the standard hydrogen electrode, is applied to first working electrodes. This value is sufficient to activate the production of protons thanks to the electrolytic solution used and to deprotect the polynucleotide fragments present in the reaction sites which comprise the first working electrodes.

For other reaction sites, where no deprotection action is to be performed on the polynucleotide fragment present because no elongation action is planned immediately, the control module may not send an instruction or send an instruction to apply a specific potential value that is known to be insufficient to activate proton production for the electrolytic solution used. In the example mentioned above, a positive potential of a value equal to 0.5 V may be applied to second working electrodes, this value being insufficient to produce enough protons to lower the pH around the sites. The polynucleotide fragments present in the reaction sites associated with these second working electrodes are thus not deprotected and they cannot consequently be elongated in the following enzymatic synthesis cycle.

As mentioned, the electrochemical device 1 of the invention is particular in that it has an array of electrodes 10 formed on a support plate 8, for example via CMOS type semiconductors, and one or more additional electrodes 16 on an opposing wall, to form field lines 17, visible in FIGS. 6 to 8, extending perpendicular to the direction of circulation of an electrolytic solution.

The at least one additional electrode 16 may be of one type or another, complementary to one or more of the working electrodes arranged on the support plate, and different arrangements of these additional electrodes 16 will be described below. In each of the cases which will be detailed below, the fact of having electrodes arranged on different walls delimiting the circulation conduit 4 of the electrolytic solution makes it possible to leave more space for the working electrodes 12 on the support plate 8 and moreover to have field lines 17 perpendicular to the direction of the flow of the electrolytic solution, as can be seen in FIGS. 6 to 8. Of course, these cases should not be considered as limiting the invention since working electrodes 12 are arranged in an array of electrodes 10 on one side of the circulation conduit 4 and one or more additional electrodes 16 are arranged on a separate wall, at a distance from the working electrodes, and for example opposite the other side of the circulation conduit 4.

The arrangement according to the invention makes it possible to have on the second wall at a distance from the support plate 8 and in particular from the CMOS chip, and where appropriate in opposition to the support plate, a reference counter-electrode large enough to compensate for each of the sets of working electrodes present on the CMOS chip. This arrangement also makes it possible to have a reference counter-electrode which is equidistant from each of the sets of working electrodes. Finally, this makes it possible to keep the species which are generated on the surface of the reference counter-electrode at a distance from the CMOS chip.

As previously mentioned, FIG. 6 illustrates an additional electrode formed by a single counter electrode, in the form of a plate. This FIG. 6 illustrates both the possibility of having an arrangement with working electrodes on the first wall 6 and one or more counter electrodes on the second wall 7, and the possibility of having a single additional electrode 16, in the form of a plate extending over a dimension substantially equal to that of the electrode array 10. It should be understood that these two characteristics could be implemented independently of each other.

As previously mentioned, FIG. 7 illustrates an additional electrode 16 formed by reference counter-electrodes 18, 20 arranged in a matrix and an electrode network 10 formed by subassemblies 19 formed by two concentric working electrodes 12. This figure also illustrates the possibility of having an arrangement with subassemblies 19 of working electrodes 12 on the first wall 6 and reference counter-electrodes 18, 20 specifically arranged on the second wall 7 opposite of each subassembly 19, that the possibility of having additional electrodes in the form of a matrix arrangement extending over a dimension substantially equal to that of the electrode network 10.

Of course, other arrangements can be implemented without departing from the context of the invention, and Figures 10 and 11 aim to expand the number of examples of arrangement to generalize on the inventive concept of the invention, namely having electrodes of a set of electrodes which are distributed on opposite walls of a circulation conduit crossed by an electrolytic solution.

Figure 9 schematically illustrates a second wall of an electrochemical device according to the invention on which several possible arrangements of additional electrodes are represented. It should be understood that several different arrangements are represented on the same intersected wall, but that in practice, one or other of the arrangements can be chosen to reproduce it over the extent of the second wall.

From left to right in this figure 9, we can thus identify two arrangements with counter-electrodes 18 alone, two arrangements with counter-electrodes combined with reference electrodes, an arrangement with reference electrodes alone, and two arrangements with counter-electrodes combined with reference electrodes.

The two arrangements with the counter electrodes 18 alone differ from each other by the size of the counter electrodes which can in particular be dependent on the size of the working electrodes 12.

Both arrangements of counter electrodes 18 combined with reference electrodes 20 have a similarity with one type of electrode being surrounded by the other type of electrode, the arrangements differing from each other in the type of electrode being surrounded.

The arrangement with reference electrodes alone is illustrated as for the other arrangements with ring-shaped electrodes, without this being limiting of the invention either for this arrangement or for the others shown in Figure 9.

Finally, the two arrangements of the counter electrodes 18 combined with reference electrodes 20, with these electrodes of two types which are concentric forming sub-assemblies 21, differ from each other in that in one arrangement the sub-assemblies 21 are formed by a reference electrode 20 which surrounds a counter electrode 20. electrode 18 while in the other arrangement the subassemblies 21 are formed by a counter electrode 18 which surrounds a reference electrode 20.

As mentioned, the additional electrodes 16 all have an annular shape here, but they could all, or some, take another shape without departing from the context of the invention, for example a square, circular, rectangular, or straight line shape. As mentioned previously, each additional electrode can also play the role of counter electrode, on the surface of which species are deposited, and of reference electrode, with a polarization pushed to a value making it possible to compensate for the currents applied to the working electrodes while maintaining a stable internal potential.

Figure 10 schematically illustrates a first wall of an electrochemical device according to the invention. Several possible arrangements of working electrodes 12 of an electrode array 10 are shown, alone or in combination with electrodes of another type. As previously, it should be understood that several different arrangements are shown on the same intersected wall, but that in practice, one or other of the arrangements can be chosen to reproduce it over the extent of the first wall.

From left to right in this figure 10, we can thus identify three arrangements with working electrodes 12 alone, three arrangements with working electrodes 12 combined with one or more reference electrodes 20, and two arrangements with working electrodes arranged concentrically.

The three arrangements with the working electrodes 12 alone differ from each other either by the position of the electrodes, with an arrangement in which a central area is free and devoid of working electrode, or by the electrical connection of the electrodes via the CMOS and the logic gates, the third arrangement comprising first working electrodes 121 and second working electrodes 122 which are capable of being supplied by different electrical potentials.

The central electrode-free area can in particular be used as a reference point for aligning the detectors of optical detection devices, such as microscopes or scanning spectrophotometers, without this list being exhaustive. The two arrangements of working electrodes 12 with one or more reference electrodes 20 differ in the number of electrodes of each type, with each time a single reference electrode in a central position, and with again a second arrangement comprising first working electrodes 121 and second working electrodes 122 which are capable of being supplied by different electrical potentials.

It should be noted that in these two arrangements, the reference electrode is larger than each of the working electrodes 12 taken respectively. This can make it possible to ensure that the surface area of the counter electrodes 18, forming the additional electrodes 16 present on the second wall 7 opposite the first wall, is greater than the surface area of the working electrodes in order to have electrochemical control of the reactions.

The arrangement with concentric reference electrodes and working electrodes is special here in that the reference electrodes 20 respectively surround the working electrode 12.

Finally, the two arrangements of the concentrically arranged working electrodes differ in the position of the first working electrodes 121 relative to the second working electrodes 121. In the first arrangement, the subassemblies are arranged alternately with a subassembly in which the first working electrode 121 surrounds the second working electrode 122 being surrounded by subassemblies in which the first working electrode 121 is surrounded by the second working electrode 122, and vice versa. In the second arrangement, the subassemblies all have the same configuration with the first working electrode surrounding the second working electrode, and a second working electrode is arranged alone between the subassemblies.

As mentioned for the second wall and the additional electrodes 16, the electrodes of the electrode network 10 all have an annular shape here, but they could all, or some, take another shape without departing from the context of the invention, for example a square, circular, rectangular, or straight line shape.

As may have been discussed with reference to FIG. 2, the additional electrode may be single and extend over the second wall over a similar extent to that of the electrode array on the first wall. In this previous example, it was mentioned that the additional electrode has a plate shape, but the additional electrode, whether formed by a counter electrode or a reference electrode, may have other forms in this context.

Figure 11 illustrates an alternative embodiment in which the additional electrode 16 has a grid shape and Figure 12 illustrates an alternative embodiment in which the additional electrode has a spiral shape.

Figure 13 illustrates another electrode arrangement variant, which can be implemented both at the first wall in the electrode array and at the second wall, provided that two distinct types of electrodes are provided. A first type of electrode, selected from a reference electrode 16, a counter electrode 18 or a working electrode 12, whether it is also a first working electrode 121 or a second working electrode 122, takes a circular shape and a second type of electrode, selected from the same list but different from the first type, takes a grid shape.

Using a grid or a spiral makes it possible to occupy the majority of the second wall 7 opposite the support plate without manufacturing complications, and thus makes it possible to optimize the performance of electrochemical reactions while reducing manufacturing costs.

We will now describe particular arrangements of the at least one additional electrode present on the second wall of the circulation conduit in opposition to the network of electrodes, these arrangements relating to the level of insertion of the additional electrode into the second wall, in particular with reference to figure 14.

As with Figures 10 and 11, it should be understood that several different arrangements are shown on the same intersected second wall, but that in practical terms, one or other of the arrangements will be chosen to reproduce it over the extent of the second wall.

Each additional electrode 16 may protrude from the second wall 7.

Each additional electrode 16 can be embedded in the second wall, in a well 22 formed in the thickness of the second wall and the depth of which is such that the additional electrode is flush with the surface of the second wall 7 delimiting the circulation conduit 4. Each additional electrode 16 can be embedded in the second wall 7, in a well 22 formed in the thickness of the second wall 7 and the depth of which is such that the additional electrode is completely retracted into the thickness of the second wall.

In a variant illustrated on the right of Figure 14, the additional electrodes can take an alternating arrangement with, at least for a given direction, one additional electrode out of two being embedded and the other additional electrode protruding from the second wall.

As described, the circulation conduit 4 is delimited by the first wall 6 and the second opposite wall 7, each being equipped with at least one electrode of the electrode assembly 2 of the electrochemical device 1. The circulation conduit 4 may contain a semi-permeable membrane 24, as illustrated in FIG. 5, or alternatively, as schematically illustrated in FIG. 2 for example, contain no membrane, and be configured only to receive a flow of liquid, or electrolytic solution, with nothing other than a vacuum between the facing electrodes. However, and in particular in a device architecture with a counter electrode formed on the wall opposite the support plate on which one or more working electrodes are arranged, in order to ensure that the species generated on the surface of the counter electrode do not reach the surface of the CMOS chip present on the support plate, it may be necessary in the absence of an ion separating membrane to provide a sufficient height between the support plate and the opposite wall.

Figure 15 illustrates a device 1 with two circulation conduits 4. These conduits have two opposing walls among which the first wall 6 defined at least in part by the support plate 8 and the second wall 7, and with the working electrodes which are arranged on the first wall 6 and the additional electrode(s) 16, whether a counter-electrode and/or a reference electrode, which are arranged on the second opposite wall 7. A semi-permeable membrane 24 is also present in the conduit and allows two different electrolytic solutions to pass through the conduit.

The electrode network 10 present on the first wall 6 comprises working electrodes 12, here nine in number, it being understood that this may not be representative of the total number of electrodes, in particular in the case of a microelectrode network, and other working electrodes 32 which are arranged respectively around one of the working electrodes 12. In this example, each electrode of the electrode network 10 is annular and a working electrode 12 and a working electrode 32 form a subset 19 of concentric annular electrodes. In this example, the working electrode 12 is electrically energized so as to function as an anode, and the working electrode 32 forms a cathode or an anode.

In this example, the additional electrode(s) 16 are reference electrodes 20 or counter electrodes 18 and they form a matrix here, on a wall at a distance from the CMOS chip on which the electrode array 10 is arranged. In accordance with what has been mentioned previously, the reference electrodes and the counter electrodes are advantageously connected to the motherboard, independently of the electrode array 10, it being understood that a connection to the CMOS chip would be possible.

It follows from the foregoing description that the invention, through all the embodiments presented, meets the aim it had set for itself, namely to provide an electrochemical device with a set of electrodes whose arrangement is different from what is known and allows easier implantation and more effective electrochemical action.

Examples

In the following examples, a solution comprising a redox couple derived from hydroquinone and benzoquinone is used to study the oxidation-reduction reaction occurring at the electrodes of different systems. These two species form a redox couple conventionally used in electrochemistry.

Among the exemplified systems, there are:

System A (Figure 16): a prior art system using two concentric platinum annular electrodes, the outer electrode being the reference counter electrode 18, 20 and the inner electrode the working electrode 12;

System B (figure 17): a second prior art system using the same electrodes as the previous system A but with the role of the electrodes reversed; thus, the reference counter-electrode 18, 20 is the internal electrode and the working electrode 12 is the external electrode;

System C (figure 18): a third prior art system using the same configuration as system B with in addition a platinum reference electrode 20 at a distance and upstream of the working electrodes 12, the counter electrode 18 therefore does not have the role of the reference electrode in this case (for example that described in US 2022/403436 A); and

System D: an exemplary system according to the present invention with an electrode array 10 composed of pairs of concentric annular platinum electrodes (like that of system A, each pair constituting a pixel) and an additional electrode 16 opposite the electrode array 10 in the form of a thin plate (we will speak of a “ceiling electrode”).

The system D can be connected in two different configurations: so-called “two-electrode” configuration (Figure 7) where the two annular electrodes of each pixel are working electrodes 121, 122 and the ceiling electrode is a reference counter-electrode 18, 20; and so-called “three-electrode” configuration (Figure 6) where the internal annular electrode of each pixel is a working electrode 12, the external electrode a counter-electrode 18 and the ceiling electrode a reference electrode 20.

Figures 19 to 26 show cyclic voltammetry curves recorded at a working electrode 12 of the different systems. The potential applied to the working electrode 12 is represented on the abscissa and the current flowing through it on the ordinate. Each voltammogram is acquired by applying, from the open-circuit potential OCP, a potential that increases progressively until a peak is observed and so as to exceed it, then the current is reduced, returning to the OCP before taking negative values (left part) and this beyond the observation of a negative peak. The absolute current value is then reduced until returning to the OCP and beyond. The shape of the voltammogram provides information on the occurrence of reactions at the working electrode 12. Thus, each peak or wave corresponds to a reaction that occurs.

In this case, it is assumed that a labile protective group is used, which is released when a sufficiently acidic pH value is reached. Thus, the solution injected into the circulation line 4 is formulated to have a more basic pH.

So, it is the positive part that interests us here. The observed peak corresponds to an oxidation reaction that causes the oxidation of the hydroquinone leading to the release of protons at the working electrode 12. These protons will then react with the protective group leading to the deprotection of the DNA fragments. The higher the amplitude of the peak, the greater the quantity of proton released. The negative part of the curve gives an idea of what is happening at the counter electrode 18 or the reference counter electrode 18, 20. In the measurement, when a negative potential is applied during the second part of the curve acquisition, the working electrode 12 actually plays the role of the counter electrode 18, where the benzoquinone reduction reaction occurs. Thus, the observed peak corresponds to this reaction which, in the real case of a deprotection step, takes place at the counter electrode 18.

The potential difference between the two peaks gives an indication of the energy required for the redox reaction to occur.

More particularly: Figure 19 shows such a curve for system A described above; Figure 20 for system B, Figure 21 for system C, Figure 22 for two-electrode system D in which the ceiling electrode is ITO; Figure 23 for three-electrode system D in which the ceiling electrode is ITO; Figure 24 for two-electrode system D in which the ceiling electrode is gold; Figure 25 for three-electrode system D in which the ceiling electrode is gold; and Figure 26 for two- or three-electrode system D in which the ceiling electrode is platinum.

Compared to gold, a higher thickness of platinum is used in this example, which helps compensate for the lower conductivity of platinum.

The table below gives the coordinates of the remarkable points, i.e. the points corresponding to the oxidation peak and the reduction peak (the values given are approximate).

The table shows that the gold and platinum ceiling electrodes are the least energy-consuming since the potential difference between the two peaks is less than 1 V in these cases. This is also the case for system C. However, the table also shows that unlike system C which requires three different types of electrodes, using a gold or platinum ceiling electrode allows the same performance to be achieved with a 2-electrode configuration. However, a 2-electrode configuration is simpler to manufacture given the less complex connections.

Regarding figures 19 and 20, a significant current difference is observed at the OCP (see box E) between the start and the time of return to this potential during voltammetry. This reflects the occurrence of a secondary reaction at the working electrode 12. In real conditions for carrying out deprotection, this means that this secondary reaction takes place in the potential range used. However, the product resulting from this reaction can pollute the oxidation procedure by reacting with the reagents, for example by reacting with the protons produced; the protons are then no longer available to react with the protective group and the fragments cannot be elongated during the subsequent elongation step. This secondary reaction can involve a radical process likely to degrade the electrode or the solution, in particular the DNA.

In comparison, the curves of the other figures return to almost 0 A upon returning to the OCP. Thus, in real conditions for carrying out deprotection, the secondary reaction does not occur or has very little chance of occurring at the working electrode 12. Thus, the desired oxidation reaction is not polluted. This allows better control of the reactions and consequently of the oligonucleotide synthesis process of which deprotection is a part.

This does not mean, however, that the secondary reaction does not take place throughout the system. On the contrary, as shown in boxes E of Figures 21 to 25, it can be guessed at on the negative side of the curve. Thus, this reaction does not occur at the electrode working electrode 12, but rather of the counter electrode 18 or of the reference counter electrode 18, 20.

However, if the counter electrode 18 or the reference counter electrode 18, 20 is arranged upstream of the working electrodes 12 in the circulation conduit 4, as in system C, the species produced by the secondary reaction can pollute the working electrodes 12 downstream.

Under the conditions for carrying out the deprotection, with regard to the two-electrode configuration of the invention, this means that the reaction takes place at the ceiling electrode 16. In this case, if there is too great a risk that the species produced by the secondary reaction at the ceiling electrode 16 will migrate to the working electrodes 12 and disrupt the oxidation reaction expected there, two solutions are possible. In a first solution, the reference counter-electrode 18, 20 can be connected to ground, which makes it possible not to polarize this electrode. A constant potential must then be applied to the working electrode 12 and the OCP can be set by the user to prevent the secondary reaction. In a second solution, a semi-permeable membrane 24 in the circulation conduit 4 between the ceiling electrode 16 and the working electrodes 12 can be provided to prevent this migration as shown in FIG. 15.

On the other hand, the secondary reaction seems to have disappeared for the system of the invention with a ceiling electrode 16 in platinum (see box E). Indeed, the wave observable in the other figures is not identifiable in the curve of figure 26.

Of course, the invention is not limited to the examples which have just been described and numerous adjustments can be made to these examples without departing from the scope of the invention.

Claims

1. Electrochemical device (1) for enzymatic synthesis of polynucleotides, comprising at least one circulation conduit (4) configured to receive an electrolytic solution, the circulation conduit (4) being delimited by a first wall (6) and a second wall (7) distinct from each other, the first wall (6) forming a support plate (8) for at least one reaction site configured for enzymatic synthesis and capable of carrying an initiator fragment of polynucleotide, in particular RNA or DNA, the circulation conduit (4) being equipped with a set of electrodes (2) controlled by a control module (14), the set of electrodes (2) comprising at least one electrode of an electrode network (10) formed on the support plate (8) and operationally associated with the reaction sites, the set of electrodes (2) also comprising at least one additional electrode (16) arranged on the second wall (7) at a distance of the support plate (8). 2. Electrochemical device (1) according to the preceding claim, in which the first wall (6) and the second wall (7) are opposite. 3. Electrochemical device (1) according to the preceding claim, in which the first wall (6) and the second wall (7) are spaced apart by a distance of 10 pm to 1 cm. 4. Electrochemical device (1) according to any one of the preceding claims, wherein said at least one circulation conduit (4) consists of one conduit or two conduits. 5. Electrochemical device (1) according to any one of the preceding claims, in which the support plate (8) carries several reaction sites, each reaction site being operationally associated with at least one electrode of the electrode network (10). 6. Electrochemical device (1) according to any one of the preceding claims, wherein the support plate (8) is configured to support at least one semiconductor, the electrode array (10) being arranged on said semiconductor. 7. Electrochemical device (1) according to any one of the preceding claims, in which the electrode network (10) is configured so as to comprise at least one working electrode (12) operationally associated with a reaction site and controlled by the control module (14) to form an anode or a cathode capable of carrying out an oxidation-reduction operation. 8. Electrochemical device (1) according to any one of claims 1 to 6, in which the electrode network (10) is configured so as to comprise several working electrodes (12) which are respectively operationally associated with a reaction site and controlled by the control module (14) to form an anode or a cathode capable of carrying out an oxidation-reduction operation, each of the working electrodes (12) being able to be controlled independently of the others. 9. Electrochemical device (1) according to any one of claims 7 or 8, in which the set of electrodes (2) comprises two distinct types of electrodes including one or more working electrodes (12) and one or more counter-electrodes (18) associated with the working electrodes (12), the at least one additional electrode (16) being an electrode of the counter-electrode type (18). 10. Electrochemical device (1) according to the preceding claim, in which the additional electrode (16) is not polarized during operation. 11. Electrochemical device (1) according to any one of claims 7 or 8, in which the set of electrodes (2) comprises three distinct types of electrodes including one or more working electrodes (12), one or more counter-electrodes (18) associated with the working electrodes (12), and at least one electrode of the reference electrode type (20). 12. Electrochemical device (1) according to the preceding claim, in which the at least one additional electrode (16) is an electrode of the reference electrode type (20) and/or an electrode of the counter-electrode type (18). 13. Electrochemical device (1) according to any one of claims 11 or 12, in which the electrode network (10) comprises several electrodes of the working electrode type (12) and of the counter-electrode type (18), an electrode of the first type being electrically supplied so as to form a cathode or an anode and surrounding at least one electrode of the second type electrically supplied so as to form an anode or a cathode. 14. Electrochemical device according to any one of the preceding claims in combination with claim 6, in which the electrode network (10) formed on the support plate (8) comprises a plurality of electrodes, the electrochemical device comprising on the second wall a single additional electrode, common to the plurality of electrodes of the working electrode type. 15. An electrochemical device according to any one of claims 1 to 13 in combination with claim 6, wherein the electrode array (10) formed on the support plate (8) comprises a plurality of electrodes, the electrochemical device comprising on the second wall (5) a plurality of additional electrodes (16) respectively associated with at least one of the electrodes of the plurality of working electrode type electrodes (12). 16. An electrochemical device (1) according to any preceding claim, wherein the working electrode (12) is supplied such that the supply current has a value greater than a threshold value, such that the working electrode (12) is made active for the production of protons or by the depletion of protons. 17. Electrochemical device according to any one of the preceding claims, in which the additional electrode (16) projects from the second wall (7) of the circulation conduit (4). 18. Electrochemical device according to any one of the preceding claims, in which the additional electrode (16) is arranged in a recess in the second wall (7) of the circulation conduit (4). 19. Electrochemical device according to any one of the preceding claims, in which the circulation conduit (4) comprises at least one semi-permeable membrane (24). 20. A method for synthesizing polynucleotides using the electrochemical device (1) according to any one of the preceding claims, comprising an association step, during which each reaction site of a support plate is operationally associated with an electrode of the electrode array, and a deprotection step (102) during which a labile protection group of a polynucleotide fragment previously formed on a reaction site is removed by an oxidation-reduction operation carried out in contact with an electrode of the electrode array, the deprotection step (102) comprising an application of an appropriate potential to at least one electrode of the electrode array (10). 21. Synthesis method according to the preceding claim, in which the deprotection step (102) and an elongation step (101), during which a nucleotide is added to the polynucleotide fragment from which the labile protection group was removed during the deprotection step, are repeated until a polynucleotide of interest is obtained. 22. A method of synthesis according to any one of claims 20 or 21, wherein the labile protecting group is sensitive to a change in pH, the application of the potential difference of the deprotection step (102) resulting in a pH change leading to cleavage of the labile protecting group. 23. The synthesis method of claim 22, wherein the protecting group is an amino group. 24. Synthesis method according to one of claims 20 to 23, in which the addition of a nucleotide to the polynucleotide fragment during the elongation step (101) is carried out by a polymerase, preferably a polymerase without a template.