Kit and method for lysis of organic elements from a biological sample by microwaves

Abstract

The invention relates to a kit for lysing organic elements of a biological sample by microwaves comprising:- a capsule (1) arranged to receive a biological sample,- at least one pair of electrodes (31, 33), and- a base (29) arranged to hold the electrodes (31, 33) of each pair of electrodes (31, 33) in respective relative positions in which the electrodes (31, 33) define a housing (35) suitable for detachably receiving a portion of the capsule (1), the base (29) and the at least one pair of electrodes (31, 33) then together forming a lysis support (3). The lysis support (3) makes it possible to establish, in the presence of an external microwave source emitting microwaves at a frequency between 0.3 and 30 GHz, an electric field having an electric force between 2 and 100 kV.m-1 within the portion of the capsule (1) received in the housing (35). [Fig. 1]

Description

Title of the invention: Kit and method for lysis of organic elements of a biological sample by microwaves

[0001] The field of the invention relates to the lysis of organic elements of a biological sample by microwaves, in particular for the purposes of testing by amplification of nucleic acids.

[0002] Molecular biology is a branch of biology that studies the properties of living beings through the structure and interactions of the molecules that compose them, and in particular biological macromolecules such as proteins, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0003] Among nuclear biology techniques, polymerase chain reaction (better known by the English acronym PCR for "polymerase chain reaction") makes it possible to produce, in vitro, a large number of copies of a DNA fragment from a low-abundance biological sample. PCR consists first of all in mixing the biological sample with nucleotides, DNA primers specific to the target fragment and a DNA polymerase, that is to say an enzyme capable of recognizing the DNA primers and assembling the nucleotides to copy the target fragment; the mixture obtained is then subjected to rapid temperature variations.In particular, the mixture is subjected to several cycles, each comprising three steps: denaturation (approximately 94-95°C) to separate the DNA strands, annealing (at a variable temperature, typically between 40 and 65°C) to attach primers to the DNA fragments, and finally, elongation (approximately 72°C) to attach DNA polymerase to the DNA primers and assemble the nucleotides.

[0004] PCR facilitates the detection of genetic material from small amounts of biological samples, which is advantageous in a medical context, for example for establishing a diagnosis. For example, a PCR test can be used as a diagnostic test for SARS-CoV-2, which is the virus responsible for Covid-19.

[0005] Nucleic acid amplification techniques such as PCR often require the prior extraction of DNA from the biological sample to be analyzed. To do this, it is known to use lysis, that is to say the destruction or decomposition of organic elements such as tissues, cells or microorganisms, and more particularly their membranes which retain the macromolecules, by physical, chemical or biological agents. Generally speaking, lysis makes it possible to release the desired chemical species - or analytes - from the organic elements.

[0006] At the same time, the recent growth in point-of-care tests (POCT) and self-tests is based on the development of simple, compact and rapid technologies.

[0007] As part of this development, several lysis processes were thus considered and tested.

[0008] Heat shock lysis involves repeating freezing / thawing cycles of biological samples to weaken and then destroy the cell membrane. However, such lysis has the disadvantage of being slow and not very effective for certain cells whose membrane is particularly resistant, such as spores or Gram-positive bacteria.

[0009] Chemical lysis involves using a detergent or chaotrope to destroy the cell membrane without denaturing or degrading the nucleic acids. However, again, such lysis is both slow and ineffective for the most resistant cells.

[0010] Mechanical lysis involves using techniques such as sonication, homogenization, or cell crushing to break the cell membrane. In particular, a process called "beat beating" involves using ball mill homogenizers, which involve grinding balls, which can be made of ceramic, glass, or stainless steel, housed in ball tubes. However, such lysis is complex to perform and requires bulky equipment.

[0011] Finally, microwave lysis involves exposing biological samples to microwaves to destroy the cell membrane. Such lysis has the disadvantage that it does not work with liquid samples with too low a conductivity and, moreover, it is necessary to precisely control the duration of exposure to microwaves as well as their frequency to avoid denaturation of the nucleic acids.

[0012] Microwave lysis is nevertheless considered promising and, consequently, innovations have been proposed to make it more effective. One possible solution, for example, involves using a "consumable," i.e., a container intended to be degraded by use and therefore to be replaced, to place the biological sample in. Such a consumable incorporates electrodes to focus the microwaves on the biological sample, which is then caught in an electric field powerful enough to destroy the cell membrane.

[0013] As examples, US patents US 9,500,590 B2 and US 10,294,451 B2 propose using a substrate on which a first and a second triangular metal structures are positioned and oriented such that a vertex of the first triangular metal structure is opposite a vertex of the second triangular metal structure. The substrate region between the two vertices forms a reaction zone at which microwave lysis can be performed.

[0014] Such a solution, however, has many drawbacks: the metals used to manufacture the electrodes release substances which are likely to inhibit subsequent PCR; only the region confined by the electrodes is affected by lysis, which represents a small volume, particularly for a liquid sample; and, in addition, a consumable incorporating electrodes is difficult to produce and is expensive for the user.

[0015] It is known, to overcome these difficulties, to use dimensional filtration to confine the biological sample in a reduced volume. The filter used is however very often oversized and its use requires several complex manipulations. The use of an extraction-purification column comprising a silica filter is also not feasible given that it does not prevent the release of PCR inhibitory substances, that it is complex to use and, finally, that it requires solutions to desorb the nucleic acids adsorbed by the silica filter.

[0016] The present invention improves the situation.

[0017] In this respect, the invention relates to a kit for lysis of organic elements of a biological sample by microwaves comprising: - a capsule designed to receive a biological sample and having a wall with a thickness of between 0.1 and 5 mm, - at least one pair of electrodes, and - a base arranged to hold the electrodes of each pair of electrodes in respective relative positions in which the electrodes are separated by a distance of between 0.3 and 20 mm so as to define a housing suitable for detachably receiving a portion of the capsule, the base and the at least one pair of electrodes then together forming a lysis support.

[0018] The lysis support makes it possible to establish, in the presence of an external microwave source emitting microwaves at a frequency of between 0.3 and 30 GHz, an electric field having an electric force of between 2 and 100 kV.m 1 within the portion of the capsule when the latter is received in the housing.

[0019] In one or more embodiments, the capsule contains an electrolyte having a conductivity of between 10 19 S.m1 and 103 Sm '.

[0020] Advantageously, the electrolyte has a conductivity of between 1 Sm 1 and 103 S.m1

[0021] In one or more embodiments, the capsule has a wall whose thickness is between 0.5 and 1.5 mm.

[0022] In one or more embodiments, the base is arranged to hold the electrodes of each pair of electrodes in respective relative positions in which the electrodes are separated by a distance of between 0.5 and 10 mm.

[0023] In one or more embodiments, the electrodes of each pair of electrodes have an elongated shape extending in a respective longitudinal direction, and the base is arranged to hold the electrodes of each pair of electrodes in respective relative positions in which the respective longitudinal directions of the electrodes form an angle of between 5° and 180°.

[0024] In one or more embodiments, the base is arranged to hold the electrodes of each pair of electrodes in respective relative positions in which the respective longitudinal directions of the electrodes form an angle substantially equal to 180°.

[0025] In one or more embodiments, the kit further comprises a plurality of spacers, each spacer being arranged to secure an electrode of the at least one pair of electrodes and the base, the electrode and the secured base being separated by a distance of between 0.1 and 80 mm, and preferably of between 0.1 and 20 mm.

[0026] In one or more embodiments, the lysis support makes it possible to establish, in the presence of an external microwave source emitting microwaves at a frequency of between 0.3 and 30 GHz, an electric field having an electric force of between 5 and 50 kV.m 1 within the portion of the capsule when the latter is received in the housing.

[0027] The invention also relates to a method for lysing organic elements of a biological sample by microwaves implemented using the kit described above. The method comprises the following operations: - place a biological sample in the capsule, - assemble the base and at least one pair of electrodes to form the lysis support, - place the capsule portion in the housing, and - expose the lysis support to an external microwave source emitting microwaves at a frequency between 0.3 and 30 GHz.

[0028] Other characteristics, details and advantages will appear on reading the detailed description below, and on analyzing the attached drawings in which:

[0029] [Fig-1] illustrates a perspective view of a capsule and a lysis support;

[0030] [Fig.2] illustrates a sectional view of an example of a capsule closed by a stopper;

[0031] [Fig.3] illustrates the cap of the capsule of [Fig.2];

[0032] [Fig.4] illustrates a side view of a pair of electrodes defining a housing suitable for receiving a capsule;

[0033] [Fig.5] illustrates a top view of the pair of electrodes of [Fig.4];

[0034] [Fig.6] illustrates a sectional view of a capsule and a lysis support;

[0035] [Fig.7] illustrates a configuration in which the lysis support comprises a single pair of electrodes and a configuration in which the lysis support comprises several pairs of electrodes;

[0036] [Fig.8] illustrates a process for lysing organic elements from a biological sample by microwave implemented with the capsule and the lysis support of [Fig.l];

[0037] [Fig.9] illustrates a process for lysing organic elements from a biological sample by microwave implemented with the capsule and the lysis support of [Fig.6];

[0038] [Fig. 10] shows the results of different PCR tests, one of which was carried out after lysis carried out with the lysis medium;

[0039] [Fig. 11] is a schematic illustration of the impedances induced by the capsule and the lysis support; and

[0040] [Fig. 12] is a photograph of a capsule and a lysis support.

[0041] [Fig.l] illustrates a capsule 1 and a lysis support 3.

[0042] Typically, the capsule 1 and the lysis support 3 are in the form of separate parts intended to be assembled, which is the case in [Fig.l] where the capsule 1 is received in the lysis support 3.

[0043] It should be noted that [Fig.l] is provided with a system of orthogonal axes X, Y and Z. The same applies to other figures described in more detail in the remainder of the description.

[0044] Capsule 1 is a container arranged to receive a biological sample.

[0045] Such a biological sample comprises organic elements such as tissues, cells or microorganisms intended to be subjected to microwave lysis. The biological sample may be liquid, solid or take the form of a gel.

[0046] Microwave lysis involves exposing the biological sample to microwaves in order to destroy or decompose the organic elements present therein. In particular, lysis can be used to destroy the membrane of the cells in the biological sample so as to extract genetic material, in particular DNA and RNA, to carry out a PCR as part of a diagnostic test.

[0047] The capsule 1 may contain an electrolyte. The property of the electrolyte to conduct electric current promotes the establishment of an electric field within the capsule 1, and therefore the lysis of the organic elements of the biological sample.

[0048] To this end, the capsule 1 is arranged to be received at least in part and in a detachable manner in the lysis support 3.

[0049] Typically, the capsule 1 is a disposable - or consumable - container, i.e. a container intended to be used a limited number of times. The capsule 1 may for example be intended to be used only once, i.e. for a single lysis.

[0050] In the example of [Fig. 1], the capsule 1 takes the form of a tube closed by a stopper. However, the capsule 1 can take any form having a portion capable of being received in a detachable manner in the lysis support 3.

[0051] In this respect, [Fig.2] illustrates an example of a capsule 1 closed by a stopper 5. Such a capsule 1 is suitable for receiving a liquid biological sample.

[0052] The present invention essentially relates to the lysis support 3. Consequently, the capsule 1 shown in [Fig.2] is described briefly below.

[0053] The capsule 1 comprises a reservoir 7, an inlet opening 9, a collection chamber 11, an absorbent layer 13, a filter 15, a clamping ring 17, an outlet opening 19 and a discharge channel 21.

[0054] The reservoir 7 - partially shown in [Fig.2] - is arranged to receive the liquid biological sample. The reservoir 7 is fluidically connected to the collection chamber 11 by the inlet opening 9.

[0055] The capsule 1 and the reservoir 7 may be separate parts, in which case the capsule 1 and the reservoir 7 must be assembled to allow the liquid biological sample to pass from the reservoir 7 to the capsule 1, and more precisely to the collection chamber 11.

[0056] Alternatively, it is possible to deposit the liquid biological sample directly into the collection chamber 11 using a dispensing instrument such as a pipette or a micropipette.

[0057] The absorbent layer 13 and the filter 15 are placed in contact with each other in the collection chamber 11. In the example of [Fig.2], the contact between the absorbent layer 13 and the filter 15 is reinforced by means of the clamping ring 17 whose external peripheral wall is in contact with the internal peripheral wall of the collection chamber 9. The clamping ring 17 is positioned inside the collection chamber 11 so as to exert pressure on the internal peripheral wall thereof.

[0058] As mentioned previously, the capsule 1 may contain an electrolyte to promote the establishment of an electric field. The electrolyte may here be absorbed by the absorbent layer 13. The absorbent layer 13 may be made from a hydrophilic material or a cellulose material. Such an absorbent layer 13 is capable of accumulating a volume of electrolyte typically between 1 and 1000 microliters (pL).

[0059] The electrolyte can be mixed with the liquid biological sample in the reservoir 7.

[0060] The filter 15 makes it possible both to filter the liquid biological sample and to evacuate the lysis product - or lysate - from the collection chamber 11. The filter 15 can take the form of a membrane, for example made of polyethersulfone, the pores of which are of a dimension generally between 0.01 and 5 micrometers (pm). The filter 15 is interposed between the absorbent layer 13 and the outlet opening 19, which allows the liquid biological sample to pass from the collection chamber 11 to the discharge channel 21 via the outlet opening 19.

[0061] The capsule 1 has for example a flexible body which can be compressed to reduce the internal volume of the collection chamber 11 so as to evacuate the liquid biological sample from the collection chamber 11 via the absorbent layer 13 and the filter 15. Other means such as a pump can be used.

[0062] It is understood from the above that the evacuation of the liquid biological sample makes it possible to recover the lysate contained in the collection chamber 11.

[0063] The cap 5 is arranged to recover the lysate, in particular for the purposes of testing by amplification of the nucleic acids.

[0064] The stopper 5 - isolated from the capsule 1 - is illustrated in [Fig.3].

[0065] The stopper 5 has a body 23 capable of being mounted on the capsule 1. For example, the internal wall of the body 23 is threaded and the body of the capsule 1 has a complementary external thread so that the cap 5 can be screwed onto the capsule 1.

[0066] The body 23 is surmounted by a shutter 25 which makes it possible to seal the evacuation channel 21 when the stopper 5 is mounted on the capsule 1.

[0067] The body 23 has two passages 27 opposite each other to receive electrodes 31 and 33 respectively. As illustrated in [Fig.2], the electrodes 31 and 33 are thus as close as possible to the collection chamber 11 and therefore to the liquid biological sample.

[0068] Reference is again made below to [Fig.l] in which a portion of the capsule 1 is received in the lysis support 3.

[0069] The lysis support 3 is arranged to receive the capsule 3 for the purpose of carrying out a lysis of the organic elements of the biological sample contained in the capsule 1 by microwaves.

[0070] The lysis support 3 comprises a base 29 and at least one pair of electrodes.

[0071] In the example of [Fig.l], the lysis support 3 comprises a single pair of electrodes, namely the pair of electrodes formed by a first electrode 31 and a second electrode 33. However, the lysis support 3 may comprise several pairs of electrodes.

[0072] For the sake of simplification, the same reference signs 31 and 33 are used in [Fig.l] and in [Fig.2] to designate the electrodes of the pair of electrodes.

[0073] The electrodes are formed from a metallic material, for example a metal such as gold (Au), silver (Ag), platinum (Pt), rhodium (Rh), palladium (Pd), aluminum (Al), copper (Cu) or an alloy. It is also possible to use another material such as a polymer coated with one of the metals mentioned above, which can be deposited by spraying, vacuum evaporation or plating.

[0074] In order to avoid electrical discharges, and in particular electric arcs, the electrodes can be treated with a passivation or anodization process. The electrodes can have a nitride coating, for example silicon nitride (Si3N4), titanium nitride (TiN) or tantalum nitride (TaN), polytetrafluoroethylene (PTFE), epoxy resin, polyurethane, graphene or diamond-like carbon (better known by the English acronym DLC for “diamond-like carbon”)•

[0075] Typically, the base 29, the first electrode 31 and the second electrode 33 are in the form of separate parts intended to be assembled to form the lysis support 3.

[0076] The base 29 is arranged to hold the electrodes of each pair of electrodes in respective relative positions in which the electrodes define a housing suitable for detachably receiving a portion of the capsule 3.

[0077] Thus, in the example of [Fig.l], the first electrode 31 and the second electrode 33 held by the base 29 define a housing 35.

[0078] Like the electrodes, the base 29 may be formed from a metallic material, for example a metal such as gold (Au), silver (Ag), platinum (Pt), rhodium (Rh), palladium (Pd), aluminum (Al), copper (Cu) or an alloy. It is also possible to use another material such as a polymer coated with one of the previously mentioned metals, which may be deposited by sputtering, vacuum evaporation or plating.

[0079] The first electrode 31 and the second electrode 33 each have an elongated shape extending in a longitudinal direction. Thus, in the example of [Fig. 1], the first electrode 31 and the second electrode 33 both extend in the direction X.

[0080] Generally speaking, when the electrodes of a pair of electrodes of the lysis support 3 each have an elongated shape and extend in a longitudinal direction, the respective longitudinal directions of these electrodes form an angle of between 5° and 180°. In the example of [Fig. 1], the first electrode 31 and the second electrode 33 are opposite each other: their respective longitudinal directions form an angle of 180°.

[0081] The first electrode 31 and the second electrode 33 have substantially the shape of a straight block. The base 29 also has an elongated shape, extends in the X direction and also has substantially the shape of a straight block.

[0082] More particularly, the first electrode 31 and the second electrode 33 each have a face having a shape complementary to the portion of the capsule 1 intended to be received in the housing 35.

[0083] [Fig.4] and [Fig.5] illustrate side and top views respectively of the first electrode 31 and the second electrode 33. The pair of electrodes 31 and 33 is that of the lysis support 3 of [Fig.1], therefore of the configuration in which the housing 35 is suitable for receiving a capsule 1 taking the form of a tube.

[0084] As illustrated in [Fig.4], the first electrode 31 has a concave face 37 which can be seen as a fillet connecting the lower face to the upper face of the first electrode 31 in the Z direction. Similarly, the second electrode 33 has a concave face 39 which can be seen as a fillet connecting the lower face to the upper face of the second electrode 33 in the Z direction.

[0085] As illustrated in [Fig. 5], the edge between the concave face 37 and the upper face of the first electrode 31 has a radius of curvature complementary to the tubular portion of the capsule 1 intended to be received in the housing 35. Such an edge typically takes the form of an arc of a circle. Similarly, the edge between the concave face 39 and the upper face of the second electrode 33 has a radius of curvature complementary to the tubular portion of the capsule 1 intended to be received in the housing 35. Such an edge also typically takes the form of an arc of a circle.

[0086] The first electrode 31 and the second electrode 33 are held by the base 29 (not shown in [Fig.4] and [Fig.5]) such that the concave face 37 and the concave face 39 are opposite each other. The inter-electrode spacing delimited in the X direction by the concave face 37 and the concave face 39 defines the housing 35.

[0087] [Fig.4] and [Fig.5] also show certain remarkable dimensions of the capsule 1 and the lysis support 3. It should be noted, however, that these figures are not to scale.

[0088] The distance di corresponds to the distance separating each of the electrodes 31 and 33 from the capsule 1. In the example of [Fig.4] and [Fig.5], each of the electrodes 31 and 33 has a complementary face of the capsule 1 intended to be received in the housing 35, in this case the concave face 37 and the concave face 39. Due to the concavity, the gap between each of the concave faces 37 and 39 and the capsule 1 varies along the Z axis, and the distance di then corresponds to the smallest gap. The distance di must be as small as possible to allow the positioning of the capsule 1, but also to strengthen the electric field to be established within the capsule 1, and more precisely the portion of the capsule 1 received in the housing 35. The distance di is less than 1 millimeter (mm).

[0089] The distance d2 corresponds to the thickness of the capsule 1, and more precisely to the thickness of the wall of the capsule 1. The distance d2 is between 0.1 and 5 mil- meters (mm), and preferably between 0.5 and 1.5 millimeters (mm).

[0090] Finally, the distance e corresponds to the distance separating two electrodes of the same pair of electrodes - in this case the first electrode 31 and the second electrode 33. More particularly, the distance e corresponds here to the distance between the concave face 37 and the concave face 39, that is to say the minimum gap between the concave faces 37 and 39. In other words, the distance e corresponds to the length of the inter-electrode spacing, and therefore of the housing 35. The distance e has a distance of between 0.3 and 20 millimeters (mm), and preferably of between 0.5 and 10 millimeters (mm). Such a distance e is sufficiently large to avoid the formation of electric arcs between the first electrode 31 and the second electrode 33.

[0091] [Fig.6] illustrates an example of assembly of the base 29 and the pair of electrodes 31 and 33 to form the lysis support 3.

[0092] In the example of [Fig.6], the capsule 1 corresponds to that illustrated in [Fig.2]. The reservoir 7, which contains the liquid biological sample and, possibly, an electrolyte, is also present.

[0093] It should be noted that, for each of the first electrode 31 and the second electrode 33, the end closest to the capsule 1 is formed by a flat face. The electrodes 31 and 33 are received by the passages 27 of the cap 5 to be as close as possible to the liquid biological sample.

[0094] It should also be noted that an external microwave source 41 is shown in [Fig.6]. Such a representation is schematic and, in practice, the external microwave source 41 takes the form of a conventional microwave oven comprising a cavity inside which the capsule 1 as well as the lysis support 3 - the capsule 1 being received in the housing 35 - are positioned.

[0095] Such an external microwave source 41 emits microwaves at a frequency between 0.3 and 30 gigahertz (GHz), and preferably equal to 2.45 gigahertz (GHz).

[0096] In the example of [Fig.6], the lysis support 3 comprises a first pair of spacers 43 and 45 and a second pair of spacers 47 and 49.

[0097] The first pair of spacers 43 and 45 is arranged to secure the first electrode 31 and the base 29 while the second pair of spacers 47 and 49 is arranged to secure the second electrode 33 and the base 29.

[0098] To do this, the base 29 has two pairs of blind holes on its upper face while each of the electrodes 31 and 33 has a pair of blind holes on its lower face. The lower face of each of the electrodes 31 and 33 is intended to be opposite the upper face of the base 29 when the electrodes 31 and 33 are held by the base 29.

[0099] The pair of blind holes of the first electrode 31 is complementary to one pairs of blind holes of the base 29 while the pair of blind holes of the second electrode 33 is complementary to the other pair of blind holes of the base 29.

[0100] It is possible to assemble the base 29 and the first electrode 31 by first inserting the spacer 43 and the spacer 45 into one pair of blind holes of the base 29 and then positioning the first electrode 31 such that the spacer 43 and the spacer 45 also fit into the blind holes of the first electrode 31. Similarly it is possible to assemble the base 29 and the second electrode 33 by first inserting the spacer 47 and the spacer 49 into the other pair of blind holes of the base 29 and then positioning the second electrode 33 such that the spacer 47 and the spacer 49 also fit into the blind holes of the second electrode 33.

[0101] When the electrodes 31 and 33 are held by the base 29, the spacers 43, 45, 47 and 49 extend along the Z direction. Consequently, the spacers 43, 45, 47 and 49 prevent any translation of the electrodes 31 and 33 along a direction other than the Z direction. In particular, the electrodes 31 and 33 cannot translate along the X direction and therefore move closer to or further away from each other.

[0102] In addition to holding the electrodes 31 and 33, the spacers 43, 45, 47 and 49 also have the function of separating the electrodes 31 and 33 from the base 29. Typically, the spacers 43, 45, 47 and 49 make it possible to separate the electrodes 31 and 33 from the base 29 by a distance of between 0.1 and 80 millimeters (mm), and preferably of between 0.1 and 20 millimeters (mm).

[0103] The spacers 43, 45, 47 and 49 must dissipate as little as possible the energy absorbed by the electrodes 31 and 33 when the latter are exposed to the microwaves emitted by the external microwave source 4L. Such spacers 43, 45, 47 and 49 must release little heat and promote the establishment of the electric field within the capsule 1, and more precisely the portion of the capsule 1 received in the housing 35.

[0104] To do this, the spacers 43, 45, 47 and 49 must be made from a suitable material. Such a material is, for example, ceramic, polytetrafluoroethylene (PTFE), polyethylene, polypropylene, polyimide, a polyepoxide or silicone. It is also possible to use a ceramic-filled polymer.

[0105] It should be noted that it is possible to use a single spacer to hold the first electrode 31 and a single spacer to hold the second electrode 33. Conversely, it is possible to use several spacers to hold the first electrode 31 and the second electrode 33, and in particular to use three or more spacers for each.

[0106] However, it is also possible, in other embodiments, not to have spacers. For example, the electrodes 31 and 33 can rest directly on the base 29 and the translational stop can then be ensured by stops present on the upper face of the base 29. Still by way of example, the upper face of the base 29 can take the form of a tank whose rim makes it possible to limit the translation of the electrodes 31 and 33 along any direction other than the Z direction.

[0107] As mentioned previously, the lysis support 3 comprises one or more pairs of electrodes. In the examples described above, the lysis support 3 comprises only one pair of electrodes, namely the pair of electrodes formed by the first electrode 31 and the second electrode 33.

[0108] [Fig.7] shows in top view two possible configurations (a) and (b) of the lysis support 3.

[0109] Configuration (a) corresponds to the case described up to now in which the lysis support 3 comprises a single pair of electrodes formed by the first electrode 31 and the second electrode 33.

[0110] Configuration (b) corresponds to a case in which the lysis support 3 comprises several pairs of electrodes, and more precisely a first pair of electrodes 51 and 53, a second pair of electrodes 55 and 57 and a third pair of electrodes 59 and 61.

[0111] For each of the configurations (a) and (b), curved lines connecting the two electrodes of each pair of electrodes correspond to the electric field induced by these two electrodes.

[0112] As explained above, when the electrodes of a pair of electrodes have an elongated shape, these electrodes extend in respective longitudinal directions which form an angle of between 5° and 180°.

[0113] In configuration (a), the respective longitudinal directions of the first electrode 31 and the second electrode 33 form an angle of 180°.

[0114] In configuration (b), the respective longitudinal directions of the two electrodes of each pair of electrodes form an angle of 45°.

[0115] A method for lysing organic elements of a biological sample by microwaves implemented with the capsule 1 and the lysis support 3 of [Fig.l] is described below with reference to [Fig.8].

[0116] The capsule 1, the lysis support 3 and the pair(s) of electrodes, here a single pair of electrodes formed by the first electrode 31 and the second electrode 33, are in the form of a kit, that is to say spare parts intended to be assembled.

[0117] During an operation 800, the cap of the capsule 1 is opened and a dispensing instrument 63 is used to deposit a liquid biological sample into the capsule 1.

[0118] On this occasion, an electrolyte can also be deposited in the capsule 1. Alternatively, the electrolyte is already present in the capsule 1.

[0119] During an operation 810, the cap of the capsule 1 is closed.

[0120] During an operation 820, the base 29 and the pair of electrodes 31 and 33 are assembled to form the lysis support 3. The base 29 holds the first electrode 31 and the second electrode 33 in respective relative positions in which they define a housing 35.

[0121] The capsule 1 is then received in the housing 35. More precisely, a portion of the capsule 1 is received in a detachable manner in the housing 35.

[0122] During an operation 830, the assembly formed by the capsule 1 and the lysis support 3 is exposed to an external microwave source emitting microwaves at a frequency between 0.3 and 30 gigahertz (GHz), and preferably equal to 2.45 gigahertz (GHz).

[0123] To do this, such an assembly is for example placed in the cavity of a conventional microwave oven, which then plays the role of an external microwave source.

[0124] The lysis support 3 makes it possible to establish an electric field having an electric force of between 2 and 100 kV.m', and preferably of between 5 and 50 kV.m', within the portion of the capsule 1 received in the housing 35. Such an electric field makes it possible to carry out a lysis of the organic elements of the liquid biological sample contained in the capsule 1.

[0125] Exposure to microwaves thus makes it possible to obtain a lysate, which contains the genetic material, in particular DNA and RNA, extracted from the organic elements of the liquid biological sample.

[0126] During an operation 840, the cap of the capsule 1 is opened and a sampling instrument is used to collect the lysate in the capsule 1.

[0127] Finally, during an operation 850, one or more analyses of the collected lysate are carried out.

[0128] A nucleic acid amplification test such as a PCR test is, for example, carried out. PCR makes it possible to produce, in vitro, a large number of copies of a DNA fragment, which facilitates the detection of genetic material.

[0129] A method for lysing organic elements of a biological sample by microwaves implemented with the capsule 1 and the lysis support 3 of [Fig.6] is described below with reference to [Fig.9]. It is understood here that the capsule 1 used is that of [Fig.2],

[0130] Here again, the capsule 1, the lysis support 3 and the pair(s) of electrodes, here a single pair of electrodes formed by the first electrode 31 and the second electrode 33, are presented in the form of a kit.

[0131] It should be noted that, in this example, the capsule 1, the cap 5 and the reservoir 7 are spare parts which are intended to be assembled to place the assembly thus formed in the lysis support 3. Such an assembly can be seen as a capsule 1 in its own right.

[0132] During an operation 900, a dispensing instrument 63 is used to deposit a liquid biological sample into the reservoir 7.

[0133] On this occasion, an electrolyte can also be deposited in the reservoir 7. Alternatively, the electrolyte is already present in the reservoir 7.

[0134] During an operation 910, the capsule 1 is mounted on the reservoir 7. For example, the internal wall of the body of the capsule 1 is threaded and the reservoir 7 has a complementary external thread so that the capsule 1 can be screwed onto the reservoir 7.

[0135] During an operation 920, a filtration of the liquid biological sample is carried out.

[0136] To do this, the assembly formed by the capsule 1 and the reservoir 7 is first turned over to circulate the liquid biological sample, and possibly the electrolyte, from the reservoir 7 to the collection chamber 11 of the capsule 1.

[0137] The user can then compress the capsule 1, which typically has a flexible body, to squeeze the capsule 1 and thereby pass the liquid biological sample through the absorbent layer 13 and the filter 15. As illustrated in [Fig.9], the filtrate of the liquid biological sample can flow from the capsule 1 through the discharge channel 21.

[0138] During an operation 930, the cap 5 is mounted on the capsule 1.

[0139] For example, the internal wall of the body 23 of the plug 5 is tapped and the body of the capsule 1 has a complementary external thread so that the cap 5 can be screwed onto the capsule 1.

[0140] During an operation 940, the base 29 and the pair of electrodes 31 and 33 are assembled to form the lysis support 3. The base 29 holds the first electrode 31 and the second electrode 33 in respective relative positions in which they define a housing 35.

[0141] The capsule 1 is then received in the housing 35. More precisely, a portion of the capsule 1 is received in a detachable manner in the housing 35. To do this, the assembly formed by the capsule 1, the cap 5 and the reservoir 7 is turned over and then placed in the housing 35. Keeping this assembly in position is made possible by the passages 27 of the body 23 of the cap 25 within which the electrodes 31 and 33 are received.

[0142] During an operation 950, the assembly formed by the capsule 1, the stopper 5, the reservoir 7 and the lysis support 3 is exposed to an external microwave source emitting microwaves at a frequency between 0.3 and 30 gigahertz (GHz), and preferably equal to 2.45 gigahertz (GHz).

[0143] In the example of [Fig.9], this assembly is placed in the cavity of a conventional microwave oven 67, which then plays the role of external microwave source.

[0144] As mentioned above, the lysis support 3 makes it possible to establish an electric field having an electric force of between 2 and 100 kV.m ', and preferably of between 5 and 50 kV.m ', within the portion of the capsule 1 received in the housing 35. Such an electric field makes it possible to carry out a lysis of the organic elements of the liquid biological sample contained in the capsule 1 and thus to obtain a lysate, which contains the genetic material, in particular the DNA and the RNA, extracted from the organic elements of the liquid biological sample.

[0145] During an operation 960, the capsule 1, the stopper 5 and the reservoir 7 are removed from the housing 35. The capsule 1, the stopper 5 and the reservoir 7 are then disassembled to recover the capsule 1 alone. The capsule 1 contains the lysate to be analyzed.

[0146] During an operation 970, the capsule 1 is mounted on a reservoir 69 containing an elution liquid.

[0147] Like the reservoir 7, the reservoir 69 may have an external thread complementary to the internal wall of the body of the capsule 1 so that the capsule 1 can be screwed onto the reservoir 69.

[0148] Finally, during an operation 980, the assembly formed by the capsule 1 and the reservoir 69 is returned to circulate the elution liquid from the reservoir 69 to the collection chamber 11.

[0149] The elution liquid allows the lysate to be evacuated, which then flows along the evacuation channel 21 and can be deposited, for example droplet by droplet, on a sample support 71 to carry out a PCR. Such a sample support 71 is for example a cell culture plate, a microfluidic cartridge or even a tube.

[0150] [Fig. 10] shows the performance of three different PCR tests, each performed on a biological sample containing 26S ribosomal RNA. 26S ribosomal RNA is a component of ribosomes, which are the cellular complexes responsible for the translation of messenger RNA (mRNA) into proteins. In particular, 26S ribosomal RNA contributes to the formation of the catalysis site where peptide bonding between successive amino acids takes place during protein synthesis.

[0151] The graph in [Fig.10] shows the evolution of the relative fluorescence unit (better known by the English acronym RFU for “relative fluorescence unit”) as a function of the number of PCR cycles.

[0152] RFU allows to quantify the fluorescence emitted by a fluorescent marker during PCR and to evaluate the quantity of DNA amplified at each cycle of PCR. RFU is a relative measurement of fluorescence compared to a reference value, which is often defined as the fluorescence emitted by a certain amount of DNA or by a standard fluorescent agent.

[0153] As detailed above, a PCR cycle comprises three steps: denaturation (approximately 94-95°C) to separate the DNA strands, annealing (at a variable temperature, typically between 40 and 65°C) to attach primers to the DNA fragments and, finally, elongation (approximately 72°C) to attach DNA polymerase to the DNA primers and assemble the nucleotides.

[0154] The first PCR test is a direct PCR test, i.e. a test based on a PCR carried out directly on the biological sample, and therefore without lysis. The curve corresponding to this first PCR test is the one marked by circles.

[0155] The second PCR test is a test based on a PCR carried out on the biological sample after it has been treated according to the process called “beat beating” using a ball mill-homogenizer from the Precellys range marketed by the company Bertin Technologies (registered trademark). The curve corresponding to this second PCR test is the one marked by diamonds.

[0156] Finally, the third PCR test is a test based on a PCR carried out on the biological sample after it has been treated according to the lysis method shown in [Fig.8] or in [Fig.9], i.e. microwave lysis carried out using the lysis support 3. The curve corresponding to this third PCR test is the one marked by triangles.

[0157] It is found that the third PCR test gives better results than the first PCR test from 15 cycles. Furthermore, it appears that the third PCR test gives better results than the second PCR test from 24 cycles.

[0158] [Fig. 11] is a pseudo-electrical diagram which illustrates the impedances induced, on the one hand, by the portion of the capsule 1 received in the housing 35 and, on the other hand, by the base 29.

[0159] The impedance Zi induced by the portion of the capsule 1 received in the housing 35 depends on several factors such as the thickness of the wall of the capsule 1, and more precisely the thickness of the capsule 1 at the level of the housing 35, the possible presence of an electrolyte or even the distance separating the electrodes of each pair of electrodes.

[0160] The impedance Z29 induced by the base 29 also depends on several factors such as the shape, dimensions and material of the base 29, the possible presence of one or more spacers and, where applicable, the length and material of the spacers.

[0161] The confinement within the portion of the capsule 1 received in the housing 35 of an electric field having sufficient electric force to achieve lysis of the organic elements of the biological sample contained in the capsule 1 is facilitated by a choice of factors allowing the impedance Zi to be strictly greater than the impedance Z29 at the microwave frequency used. Indeed, the energy absorbed by the electrodes when they are exposed to microwaves must preferably dissipate at the level of the portion of the capsule 1 received in the housing 35, and not in the base 29 or in the spacers mechanically connecting the base 29 and the electrodes.

[0162] To do this, the Applicant carried out experiments which indicated that, in the presence of an external microwave source emitting microwaves at a frequency of between 0.3 and 30 gigahertz (GHz), and preferably equal to 2.45 gigahertz (GHz), such a condition requires at least that the thickness of the capsule 1 be between 0.1 and 5 millimeters (mm), and preferably between 0.5 and 1.5 millimeters (mm), and that the electrodes of each pair of electrodes be separated by a distance of between 0.3 and 20 millimeters (mm), and preferably between 0.5 and 10 millimeters (mm).

[0163] The electrical strength of the electric field established by the lysis support 3 within the portion of the capsule 1 received in the housing 35 is then between 2 and 100 kV.m ', and advantageously between 5 and 50 kV.m '.

[0164] In any event, and among the factors listed above, obtaining an impedance Zi greater than the impedance Z29 is guaranteed by the presence in the capsule 1 of an electrolyte having a conductivity of between 10 19 Sm 1 and 103 Sm ', and preferably of between 1 Sm 1 and 103 Sm '. An electrolyte having such conductivity has systematically enabled the Applicant to obtain an electric field having the electric force required to carry out lysis of the organic elements of the biological sample.

[0165] Of course, within the perimeter delimited by the necessary conditions mentioned above - namely the conditions relating to the thickness of the wall of the capsule 1 and to the distance separating the electrodes of each pair of electrodes, the Applicant has found that it was possible to play on several factors or parameters to obtain the electric field having the desired electric force.

[0166] In particular, in addition to the factors having an influence on the impedances Zi and Z29, it is possible to vary the number of pairs of electrodes, the angle formed by the respective longitudinal directions of the electrodes of each pair of electrodes, the shape, dimensions and material of the electrodes.

[0167] In particular, for a given frequency of microwaves emitted by the external microwave source, it is particularly advantageous for the electrodes to have a length of the order of X / 2 or X / 4, where X is the wavelength of the microwaves; and / or for the base 29 to have a length of the order of / . or X / 2.

[0168] Furthermore, whatever the embodiment envisaged, it should be noted that the establishment of an electric field having an electric force of between 2 and 100 kV.m 1 within the portion of the capsule 1 received in the housing 35 in the presence of an external microwave source emitting microwaves at a frequency between 0.3 and 30 gigahertz (GHz) can be verified directly.

[0169] Indeed, to evaluate the performance of an embodiment, the Applicant carried out the thermal test detailed below.

[0170] An electrolyte solution is poured into a standard capped tube with a capacity of 2.0 milliliters (mL). This electrolyte solution contains a Tris HCl buffer. The Tris HCl buffer has a concentration in the electrolyte solution of 250 millimoles per liter (mM).

[0171] The standard capped tube is positioned in a foam holder, which is placed in a conventional microwave oven emitting microwaves having a frequency of the order of 2.45 gigahertz (GHz). A timer starts at the same time as the conventional microwave oven. The pressure induced by the heated electrolyte solution eventually causes the cap to pop off the tube and the timer is then stopped. The time obtained - here 120 seconds (s) - constitutes a reference time.

[0172] For each lysis support 3 to be tested, the same experiment is reproduced but replacing the foam support with lysis support 3.

[0173] By way of example, [Fig. 12] is a photograph of a lysis support 3 successfully tested by the Applicant.

[0174] The comparison between the time measured for lysis support 3 and the reference time makes it possible to evaluate the performance of lysis support 3. A measured time five times lower than the reference time, therefore 24 seconds (s), highlights the establishment of an electric field having adequate electric strength.

Claims

Claims

1. Kit for lysis of organic elements of a biological sample by microwaves comprising: - a capsule (1) arranged to receive a biological sample and having a wall whose thickness is between 0.1 and 5 mm, - at least one pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61), and - a base (29) arranged to hold the electrodes (31, 33, 51, 53, 55, 57, 59, 61) of each pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) in respective relative positions in which said electrodes (31, 33, 51, 53, 55, 57, 59, 61) are separated by a distance between 0.3 and 20 mm so as to define a housing (35) suitable for detachably receiving a portion of said capsule (1), said base (29) and the at least one pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) then together forming a lysis support (3), said lysis support (3) making it possible to establish, in the presence of an external microwave source (41,67) emitting microwaves at a frequency between 0.3 and 30 GHz, an electric field having an electric force between 2 and 100 kV.m1 within the portion of the capsule (1) when the latter is received in said housing (35).,

2. A kit according to claim 1, wherein the capsule (1) contains an electrolyte having a conductivity of between 10 19 Sm 1 and 103 Sm '.

3. A kit according to claim 2, wherein the electrolyte has a conductivity of between 1 S.m1 and 103 Sm '.

4. Kit according to one of the preceding claims, in which the capsule (1) has a wall whose thickness is between 0.5 and 1.5 mm.

5. Kit according to one of the preceding claims, wherein the base (29) is arranged to hold the electrodes (31, 33, 51, 53, 55, 57, 59, 61) of each pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) in respective relative positions in which said electrodes (31, 33, 51, 53, 55, 57, 59, 61) are separated by a distance of between 0.5 and 10 mm.

6. Kit according to one of the preceding claims, in which the electrodes (31, 33, 51, 53, 55, 57, 59, 61) of each pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) have an elongated shape extending in a respective longitudinal direction, and in which the base (29) is arranged to hold the electrodes (31, 33, 51, 53, 55, 57, 59, 61) of each pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) in respective relative positions in which the respective longitudinal directions of said electrodes (31, 33, 51, 53, 55, 57, 59, 61) form an angle between 5° and 180°.

7. A kit according to claim 6, wherein the base (29) is arranged to hold the electrodes (31, 33, 51, 53, 55, 57, 59, 61) of each pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) in respective relative positions in which the respective longitudinal directions of said electrodes (31, 33, 51, 53, 55, 57, 59, 61) form an angle substantially equal to 180°.

8. Kit according to one of the preceding claims, further comprising a plurality of spacers (43, 45, 47, 49), each spacer (43, 45, 47, 49) being arranged to secure an electrode (31, 33, 51, 53, 55, 57, 59, 61) of the at least one pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) and the base (29), said secured electrode (31, 33, 51, 53, 55, 57, 59, 61) and said base (29) being separated by a distance of between 0.1 and 80 mm, and preferably of between 0.1 and 20 mm.

9. Kit according to one of the preceding claims, in which the lysis support (3) makes it possible to establish, in the presence of an external microwave source (41, 67) emitting microwaves at a frequency of between 0.3 and 30 GHz, an electric field having an electric force of between 5 and 50 kV.m1 within the portion of the capsule (1) when the latter is received in said housing (35).

10. Method for lysing organic elements of a biological sample by microwaves implemented by means of the kit according to one of the preceding claims, said method comprising the following operations - depositing (800, 900) a biological sample in the capsule (1), - assembling (820, 940) the base (29) and the at least one pair of electrodes (31, 33, 51, 53, 55, 57, 59, 61) to form the lysis support (3), - placing (820, 940) the capsule portion (1) in the housing (35), and - exposing (830, 950) the lysis support (3) to an external microwave source (41, 67) emitting microwaves at a frequency between 0.3 and 30 GHz.

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