Capsule and method for compartmentalizing extraction particles in a container

Abstract

The invention relates to a capsule comprising extraction particles for capturing the nucleic acids of a sample to be tested, said capsule comprising at least: a shell that is soluble in water and / or in a lysis buffer for the cells of the sample to be tested, the shell delimiting an internal volume, - extraction particles, preferably silica particles and more preferentially magnetic silica particles, an excipient that is soluble in water and / or a lysis buffer for the cells of the sample to be tested, said excipient also being compatible with the stability of the shell of the capsule and compatible with the performance of the extraction particles, the particles and the excipient being contained in the internal volume delimited by the shell of the capsule. The invention also relates to a container comprising said capsule and also to a method for extracting and detecting the nucleic acids from a sample to be tested using said capsules.

Description

[0001] DESCRIPTION

[0002] Title: Capsule and method for compartmentalizing extraction particles in a container

[0003] Technical field of the invention

[0004] The invention relates to the technical field of molecular biology and more particularly to systems for extracting biomolecules using extraction particles from samples containing at least one analyte. Obviously, the present invention can be applied to other technical fields to which it would be adapted.

[0005] Technological background of the invention

[0006] In extraction systems, the cells of the sample are lysed to extract their contents, particularly biomolecules (nucleic acids – DNA and / or RNA – or proteins). These biomolecules are then captured for analytical or diagnostic testing.

[0007] Cell lysis in the sample can be mechanical, chemical, or a combination of both.

[0008] For capturing biomolecules (nucleic acids and / or proteins), a different mechanical support is usually used than that used for lysis, and preferably a different type of particle. For example, silica-coated particles are used to capture nucleic acids. Preferably, these particles are magnetic and can be manipulated using a magnetic device. These magnetic extraction particles containing silica are also commonly called silica beads, for example, NucliSENS® easyMAG® Magnetic Silica (MagSIL - bioMérieux, France). For capturing proteins, several techniques can be used (electrophoresis, chromatography, precipitation). For example, a support carrying an antibody that will bind specifically to the protein of interest can be used. This could be magnetic particles, with or without silica, carrying an antibody or a molecule that binds to the protein of interest.

[0009] Extraction particles bound to lysed cell constituents generally need to be washed. Particle washing is carried out in various ways: passing through several containers filled with identical or different washing solutions with or without agitation, using discontinuous magnetization within the same container, or by agitation when the particles are magnetic.

[0010] Next, the biomolecules or molecules of interest (nucleic acids and / or proteins depending on the characteristics of the capture support used) are dissociated from the capture support or extraction particles via an elution step at the end of which the biomolecules (nucleic acids and / or proteins) contained from the sample to be tested are recovered, whether they are in the cells or organelles of the sample or in the circulating liquid phase of the sample.

[0011] Finally, after the elution phase, a nucleic acid amplification phase is generally carried out in order to obtain a sufficient quantity of nucleic acids to allow them to be detected subsequently in a rapid, sensitive and reliable manner.

[0012] During this process of extracting biomolecules from the sample to be tested, it becomes clear that the capture step is crucial for the rest of the process and in particular that the capture particles themselves are the key and crucial elements for an efficient capture of nucleic acids and / or proteins, for the maximum performance of an extraction platform or kit and more generally, ultimately, for making a reliable diagnosis.

[0013] It is therefore essential that these extraction particles be in optimal conditions to bind to biomolecules, but it is also crucial that they are all extracted, collected, and recovered after the extraction process. Indeed, once these extraction particles are collected, the bound biomolecules are eluted, followed by an amplification phase when the biomolecules are nucleic acids. The captured biomolecules are then detected to provide a diagnostic result. Collecting all or as many extraction particles as possible is essential to avoid losing key information from the captured biomolecules and thus to obtain the most reliable, accurate, complete, and rapid diagnostic test result.

[0014] Several delicate steps are involved with these extraction particles:

[0015] - They must be dispensed into a container into which the sample has been or will be introduced. This container is generally small (on the order of a few ml, usually around 2 to 3 ml), and the quantity of extraction particles per container is approximately 1 mg, representing a volume of these particles of about ten to one hundred µl, depending on their dilution. Dispensing such a small quantity and volume of extraction particles into the container can be challenging. This requires a high degree of precision, especially since these particles generally settle, and a particle mixing system is necessary to resuspend them. Therefore, achieving precise and reproducible particle distribution within the test container is difficult.This container can subsequently be referred to interchangeably as an extraction device, extraction cartridge, extraction strip, or extraction plate. The distribution of the extraction particles generally occurs during the manufacturing process of the extraction devices or cartridges that will be sold. However, in some cases, the distribution can be performed extemporaneously or directly into the analytical instrument, and this step is carried out by the client / laboratory that will perform the test or analysis. Therefore, a highly reproducible process is essential, which is not straightforward and is not currently optimal.

[0016] To dispense these extraction particles as accurately as possible, one solution would be to dilute them, but their stability could be compromised by large dilution volumes. Furthermore, the volume or size of the container / device holding them is a limiting factor for the volume of diluted particles that can be introduced, as this container must be able to hold at least the sample to be tested, the lysis buffer, the extraction particles, a stirring device, and a sampling device configured to collect the particles loaded with biomolecules. To prevent the material in the container from overflowing during the sample testing process, and also to avoid destabilizing the extraction particles, it is therefore necessary to find a system that eliminates the need to dilute the extraction particles for dispensing.

[0017] - During transport of the extraction container or cartridge containing the reagents and extraction particles, it is very likely that the container will be moved, shaken, agitated, inverted, or not remain in the same plane throughout transport, ideally horizontal (parallel to the plane of the lid, foil, or opening of the container). This occurs before the sample is added to the container, and in this case, some of the extraction particles will coat the walls of the container and / or be projected onto the walls and / or onto the lid, the container sealing film, or the foil / opening (plastic or aluminum).This is very problematic because not only is there a risk of losing some of the particles when removing the film or lid, but this also causes the dissemination of extraction particles into the analytical instrument or the analysis station if the analysis is carried out manually, and therefore its contamination and / or soiling.

[0018] Furthermore, some of the extraction particles can be projected onto the walls of the extraction vessel or the well of the extraction cartridge in which they are contained. The sample and reagent volumes may be insufficient to allow the extraction particles on the walls to be drawn back into the mixture, submerged, and thus capable of capturing the biomolecules in the sample being tested. This further reduces the biomolecule collection capacity and also impairs the reliability and reproducibility of the test. This problem of magnetic element projection is addressed in patent USS9410185. The proposed solution is to embed the elements in a film or to apply a film to the elements at the bottom of the test vessel, but there is no mention of an excipient or liquid containing the magnetic elements.This proposed solution is therefore unsuitable because it has been found that magnetic particles must not be dried, freeze-dried, or otherwise dehydrated to maintain their full performance. They must remain in an excipient, and this excipient must not dissolve or solubilize the film in which they are embedded or which holds them to the bottom of the container. The excipient must therefore not be dry or be dried out to avoid altering the performance of the magnetic particles it contains.

[0019] Once the sample lysis phase is complete and the biomolecules are bound to the extraction particles, it is necessary to collect these particles for washing. This ensures clean, purified biomolecules, free of agents that could interfere with their detection. In the case of nucleic acids, this is crucial for removing as many agents as possible that could inhibit their amplification. Collecting the extraction particles is a delicate but critical step. All particles must be collected to achieve the best biomolecule capture yield and a reliable diagnostic result. If some particles are scattered and / or splashed onto a wall, and especially if some extraction particles are outside the liquid (sample + reagents), it will be very difficult to collect them for washing before eluting the bound biomolecules.Indeed, this particle collection phase is carried out either by pipetting the liquid containing the extracted particles, or using a magnetic device or magnet that gathers or aggregates the particles, followed by pipetting this aggregate of extracted particles. Particle collection for washing can be done using only a magnetic device, followed by transferring this device to the washing area.

[0020] During the production or industrialization phases of extraction containers or cartridges, in addition to the problems described above, it is essential that the extraction particles, particularly silica-based particles, not be dried. Dried silica particles lose their biomolecule capture performance, especially their nucleic acid capture performance. This applies not only to the industrialization phase but also to the long-term storage of test containers or cartridges, which must be carried out without evaporation, at room temperature; otherwise, the performance and reliability of the test will be compromised.

[0021] The storage conditions of the extraction particles are crucial because their performance must be maintained over time, preferably at room temperature, regardless of the storage medium. In liquids, many additives present in a storage solution can adsorb onto the extraction particles during storage, degrading their binding capacity to biomolecules and thus their extraction performance, thereby altering the final test result (see WO2019175518A1). Therefore, it is essential that the particles remain stable during long-term storage and maintain their performance.

[0022] - It is also important to have precise and certain localization of the extracted particles within the container or cartridge containing them, both during transport and during the diagnostic test. This is especially true in an automated or semi-automated biomolecule extraction protocol.

[0023] Object of the invention

[0024] It therefore appears that many problems remain to be solved regarding these extraction particles, magnetic or otherwise, during the process of extracting biomolecules from the sample (also called "sample preparation"), whether performed manually or automatically. This must be resolved so that the biomolecules of interest likely to be contained in the sample can be captured, detected, and analyzed rapidly, efficiently, reliably, and reproducibly.

[0025] The object of the present invention is to improve the industrialization, distribution, storage and use processes of extraction particles contained in an extraction cartridge, to improve the stability of the extraction particles and to simplify the production process of the test container or the extraction cartridge containing the extraction particles by compartmentalization, and all with a view to obtaining a clean, efficient, optimal, reliable, rapid and reproducible extraction of biomolecules.

[0026] To this end, the invention relates to a capsule containing particles for extracting biomolecules from a sample to be tested, comprising at least: a closed envelope soluble in an aqueous buffer, a buffer for lysis of cells from the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume,

[0027] - Extraction particles of the biomolecules from the sample, preferably silica particles and more preferably magnetic silica particles, an excipient soluble in an aqueous buffer, a buffer for lysis of the cells of the sample to be tested and / or in the liquid sample to be tested, the extraction particles of the biomolecules and the excipient being contained in the internal volume delimited by the capsule shell, said excipient being compatible with the stability of the capsule shell but also compatible with the performance of the extraction particles, and the shell once solubilized being compatible with the performance of the extraction particles.

[0028] The invention also relates to a container or extraction device containing at least one capsule according to the invention.

[0029] The invention also relates to a diagnostic kit comprising at least one capsule according to the invention. The invention further relates to a method for manufacturing the capsules according to the invention, comprising the following steps:

[0030] Prepare a suspension of biomolecule extraction particles at a given concentration in the desired excipient or mixture of excipients, compatible with the nature of the coating that will be used to form the capsule.

[0031] Prepare a capsule envelope or use a pre-formed capsule envelope,

[0032] Fill the envelope of the extraction particle suspension using suitable equipment,

[0033] Seal the envelope so that it is airtight.

[0034] Finally, the invention also relates to a method for extracting biomolecules contained in the sample to be tested, comprising:

[0035] A step of bringing the lysed or unlysed sample into contact with the capsule according to the invention, the solubilized contents of said capsule, or only the extraction particles previously contained in said capsule,

[0036] A lysis step of the sample before and / or after contacting the unlysed sample and the capsule according to the invention, the solubilized contents of said capsule or the extraction particles previously contained in said capsule,

[0037] A step involving the capture of biomolecules from the sample to be tested by extraction particles,

[0038] - Optionally, a washing step for the biomolecules captured by the extraction particles,

[0039] - Optionally, an elution step of the biomolecules captured by the extraction particles.

[0040] Finally, the invention relates to a detection method which corresponds to the extraction method mentioned above and which further comprises:

[0041] - Optionally, a nucleic acid amplification step when the biomolecules extracted at the end of the extraction process according to the invention are nucleic acids; preferably, the amplification is carried out by PCR.

[0042] A step involving the detection of biomolecules using appropriate methods. These biomolecule extraction and detection processes allow for ease of distribution and handling of extraction particles, greater reliability, reproducibility of test results, and rapid testing.

[0043] Description of the figures

[0044] The invention will be better understood from the following description, which relates to an embodiment of the present invention, given by way of non-limiting example and explained with reference to the accompanying schematic figures. The accompanying schematic figures are listed below:

[0045] [Fig. 1] is a diagram of variants of the method for solubilizing the capsule of the invention in one or more containers that can be used for the extraction of biomolecules from a sample to be tested. In (A), a test container (3) is shown containing a capsule (1) according to the invention containing extraction particles and one or more excipients. The sample (2) is added to the container, which solubilizes the capsule's outer layer. This results in a container containing the sample mixed with the solubilized capsule, i.e., a mixture (4) of the sample with the solubilized outer layer, the excipient, and the extraction particles. In (B), a test container is shown containing the sample (2). The capsule (1) is added to the container, which solubilizes it.A container is thus obtained containing the sample mixed with the solubilized capsule, that is, a mixture (4) of the sample with the solubilized capsule shell, the excipient, and the extraction particles. Figure (C) shows a test container (3) containing a capsule (1) according to the invention containing extraction particles and one or more excipients. An aqueous buffer (5) or a lysis buffer of the sample (5) is added to the container, which solubilizes the capsule shell. The resulting capsule is solubilized in the aqueous buffer or lysis buffer, that is, a mixture (6) comprising said buffer with the solubilized capsule shell, the excipient, and the extraction particles. This mixture (6) is transferred into a container containing the sample (2).This results in a container comprising the sample mixed with the capsule solubilized in the aqueous buffer or lysis buffer, i.e., a mixture (7) of the sample with the aqueous buffer or lysis buffer, the solubilized capsule shell, the excipient, and the extraction particles. Figure (D) shows a test container (3) containing a capsule (1) according to the invention containing extraction particles and one or more excipients. An aqueous buffer (5) or a lysis buffer of the sample (5) is added to the container, which solubilizes the capsule shell. The resulting capsule is solubilized in the aqueous buffer or lysis buffer, i.e., a mixture (6) comprising said buffer with the solubilized capsule shell, the excipient, and the extraction particles. A sample (2) is added to this mixture (6).This results in a container comprising the sample mixed with the capsule solubilized in the aqueous buffer or lysis buffer, i.e., a mixture (7) of the sample with the aqueous buffer or lysis buffer, the solubilized capsule, the excipient, and the extraction particles. In (E), a test container containing the sample (2) is shown, to which an aqueous buffer (5) or a lysis buffer of the sample (5) is added. This has the effect of lysing the sample. Furthermore, a test container (3) is shown containing a capsule (1) according to the invention, containing extraction particles and one or more excipients. The lysate obtained at the end of the first step (mixing the sample (2) with the aqueous lysis buffer (5)) is added to the container containing the capsule. This has the effect of solubilizing the capsule shell.The capsule is thus solubilized in the lysate, resulting in mixture (7) which corresponds to the sample with the aqueous buffer or lysis buffer, the solubilized capsule shell, the excipient, and the extraction particles. Figure (F) shows a test container (3) containing a capsule (1) according to the invention containing extraction particles and one or more excipients. An aqueous buffer (5) or a lysis buffer of the sample (5) is added to the container, which solubilizes the capsule shell. The resulting capsule is solubilized in the aqueous buffer or lysis buffer, that is, a mixture (6) comprising said buffer with the solubilized capsule shell, the excipient, and the extraction particles. In this mixture (6), a magnet or magnetized device (8) is introduced which attracts the magnetic extraction particles, and this is all the more so when the magnetized device is rotated (symbolized by the arrow).The device loaded with extraction particles (9) is removed. It is then immersed in a container containing the sample (2) to which an aqueous buffer (5), or lysis buffer of the sample (5), has been added. The device is rotated again, which resuspends the extraction particles in the lysed sample (lysate). These extraction particles can then capture the biomolecules of the sample to be tested. In this embodiment, a final mixture (10) is obtained, comprising the sample, the lysis buffer, and the particles. There is no final mixture of the sample and the entire solubilized capsule. Here, the sample is not mixed with the solubilized capsule and the excipient.

[0046] Of course, Figure 1 is not exhaustive, and there are many variations not shown that are covered by the present invention. [Fig. 2] illustrates the technical problem related to extraction particles that are dispersed and projected onto the walls of the wells of a test container or biomolecule extraction device when the extraction particles are not compartmentalized / contained in a capsule according to the invention. a) corresponds to the plastic test container comprising several wells. b) corresponds to the extraction particles (50 µl of NucliSENS® easyMAG® MagSIL, bioMérieux) at the bottom of one of the container's wells. c) corresponds to the biomolecule extraction particles dispersed and projected onto the walls of the sample's test container wells during transport, preventing the complete recovery of said extraction particles and also causing them to dry against the walls.

[0047] [Fig. 3] corresponds to a diagram of a capsule according to the invention and of the process of lysis of the latter, thus releasing its contents.

[0048] (1) corresponds to the soluble envelope in an aqueous buffer, a cell lysis buffer of the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume filled by

[0049] (2) magnetic extraction particles suspended in a liquid excipient soluble in an aqueous buffer, a cell lysis buffer of the sample to be tested and / or in the liquid sample to be tested and being compatible with the stability and integrity of the capsule shell but also compatible with the performance of the extraction particles

[0050] (3) corresponds to the dispensing of the capsule according to the invention in a container or consumable dedicated to the extraction of biomolecules from a sample to be tested.

[0051] (4) corresponds to the addition of a sample lysis buffer which will at least partially solubilize the capsule shell

[0052] (5) corresponds to the extemporaneous dissolution of the envelope which releases its contents into the external environment.

[0053] [Fig. 4] corresponds to three examples of capsules according to the invention.

[0054] (A) represents a pullulan soluble capsule filled with magnetic particles suspended in the excipient glycerol at 90% by weight (w / w),

[0055] (B) represents polyvinyl alcohol (PVA) soluble sachets filled with magnetic particles suspended in the excipient glycerol at 95% by weight (w / w), (C) represents a soft ellipsoidal gelatin capsule filled with magnetic particles suspended in the excipient refined sweet almond oil, at 98% by weight (w / w) in ethanol.

[0056] Detailed description

[0057] CAPSULE

[0058] The present invention covers a capsule containing particles for extracting biomolecules from a sample to be tested, comprising at least: a closed envelope soluble in an aqueous buffer, a buffer for lysis of cells from the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume, particles for extracting biomolecules, preferably silica particles and more preferably magnetic silica particles, an excipient in liquid or viscous form, soluble in an aqueous buffer, a buffer for lysis of cells from the sample to be tested and / or in the liquid sample to be tested, the particles for extracting biomolecules and the excipient being contained in the internal volume delimited by the envelope of the capsule.

[0059] The invention also covers a capsule containing particles for extracting biomolecules from a sample to be tested, comprising at least:

[0060] - a closed envelope soluble in water, an aqueous buffer, a buffer for cell lysis of the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume,

[0061] - particles for extracting biomolecules, preferably silica particles and more preferably magnetic silica particles,

[0062] - a water-soluble excipient, an aqueous buffer, a lysis buffer for cells from the sample to be tested and / or in the liquid sample to be tested, the biomolecule extraction particles and the excipient being contained within the internal volume delimited by the capsule shell, said excipient being compatible with the stability of the capsule shell and also compatible with the performance of the extraction particles, and the shell, once solubilized, being compatible with the performance of the extraction particles. The term analyte refers to any microorganism or fragment of a microorganism (bacteria, virus, yeast), as well as nucleic acids or proteins from the host.

[0063] A capsule is defined as a solid structure, enclosing an internal space or volume at least partially filled with liquid, solid, or both compounds. The capsule may be flexible or rigid. It allows for the containment of elements and their isolation from the external environment. In the present invention, the capsule, when filled and sealed, isolates the extracted particles or compartmentalizes them within a defined space of the extraction or testing container or device.

[0064] Compartmentalization refers to the ability to protect from, keep apart from, partition, confine, isolate, or separate. In this case, the capsule is a means of compartmentalizing the space of the test container and allowing the extraction particles to be placed in a defined location and in a concentrated manner without being able to disperse on the walls of the test container and without drying out or degrading.

[0065] ENVELOPE

[0066] The term "encapsulation" refers to a closed external structure defining an internal volume that can be filled with at least one composition distinct from the composition of the encapsulation itself. The encapsulation constitutes the outer part of the capsule. It can be flexible or rigid. It is airtight and therefore continuous, even if it is made up of several parts (for example, in the case of a capsule), because the two parts are then sealed to close the encapsulation and make it airtight, preventing leakage of excipient liquid or particles. The encapsulation must not allow its contents to escape before the actual implementation of a biomolecule extraction process. It can thus be said that it must be closed.

[0067] According to the present invention, the capsule is in a solid, continuous form when intact and whole, but the capsule can also be in a solubilized, dissolved form, wholly or partially, when the capsule comes into contact with water, an aqueous buffer, a lysis buffer, and / or a liquid or partially liquid sample, lysed or not, which has the effect of releasing the capsule's contents into the surrounding medium. When the capsule is subjected to mechanical lysis, the capsule can also become fragmented, broken, and porous, thus allowing its contents to be released and dispersed into the surrounding medium.The casing used in the present invention can have any suitable composition provided that it fulfills the functional objectives described herein, namely, that it forms a barrier between the external environment and the internal environment that it delimits or defines, and that it is at least partially soluble in a defined medium, thus allowing the release of its contents (extraction particles and excipient). The casing of the invention can be formed, at least in part, from one or more known soluble substances. For example, any organic or inorganic polymeric material, or a material derived from one or more such materials, characterized by its easy solubility in the defined medium.These substances may include cellulose-based or derivative materials, such as low-viscosity cellulose, hydroxyalkylmethylcellulose (HPMC), or carboxymethylcellulose, polyvinyl alcohol (PVA), gelatin (preferably bovine, porcine, or fish gelatin), pullulan, carrageenan, casein, or a mixture of at least two of these. Other suitable materials may include a combination of hydroxyalkyl carboxylic acid ester monomers with a hydroxyalkyl carboxylic acid ester monomer and an ethoxylated or propoxylated monomer of (meth)acrylate or polyethylene glycol (PEG). Formulations containing various amounts or combinations of the aforementioned substances are also considered.

[0068] In a preferred embodiment of the invention, the capsule shell comprises polyvinyl alcohol (PVA); hydroxypropyl methylcellulose (HPMC); carboxymethylcellulose; gelatin, preferably bovine, porcine, or fish gelatin; pullulan; carrageenan; or a mixture of at least two of these. More preferably, the capsule shell is made of polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), gelatin, or a mixture of at least two of these substances. Even more preferably, the shell is made primarily of only one of these compounds and not a mixture of them.

[0069] It is obviously possible to use commercially available, pre-formed capsules, provided they contain at least one of the above-mentioned components as their main components and fulfill the properties described above. For example, empty capsules or softgels of various sizes, from 0 to 5, offered by LGA or Aromazone (France) can be used. In a preferred embodiment of the invention, the capsule shell comprises at least 80% by weight (w / w) of polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose, gelatin, pullulan, carrageenan, or a mixture of at least two of these, preferably at least 90% by weight (w / w), more preferably 95% by weight (w / w), and even more preferably 100% by weight (w / w).Unlike prior art solutions (US7419903, US9410185), it is understood that the capsule shell according to the invention does not contain breath fresheners, flavorings (which could impair the functionality of the extraction particles), or magnetically reacting particles such as the extraction particles used in the present invention. According to the present invention, the extraction particles are contained within the lumen of the capsule or the internal volume delimited by the capsule shell, but they are not incorporated into the shell itself. The prior art mentions the use of film but does not mention the use of capsules per se, and even less so capsules containing an excipient in which extraction particles are suspended.If the extraction particles were incorporated or integrated into the capsule shell, they would lose functionality and performance; that is, the particles would have more difficulty, or even none at all, capturing the biomolecules contained in the sample to be tested because they would have been dried beforehand, which is not the case in the present invention. Furthermore, the extraction particles are contained in a defined excipient chosen in such a way that the capsule shell is not solubilized or lysed by this excipient. Indeed, the capsule shell is solubilized only by a means external to the capsule and not from within. This can be a physical or mechanical means (shearing, abrasion, agitation, use of external particles such as ceramic beads or an element configured to at least weaken, crack, and rupture the shell, heating, etc.).), by a chemical means such as an aqueous buffer, a lysis buffer of the sample, by the sample itself) or by a combination of at least two of these means.

[0070] The envelope is airtight, meaning that its contents cannot escape without deliberate action. It does not leak; its liquid or viscous contents remain within the internal space or volume it defines. The shape of the envelope determines the shape of the capsule. Thus, according to one feature of the invention, the envelope, and therefore the capsule, is closed and has an ellipsoidal, spherical, cylindrical, cubic, parallelepiped, conical, pyramidal, tetrahedral, octahedral, triangular prism, rectangular or cuboid, pentagonal, hexagonal, tubular, capsule, bag, sachet, or pyramidal shape. Although all shapes are suitable for the present invention, the preferred and most commonly used shapes for practical manufacturing and handling reasons are the capsule or sachet shape.

[0071] According to the features of the invention, the capsule can exist in two forms: a solid form, filled with the excipient and extraction particles, or a dissolved form, when the capsule is solubilized by an external, deliberate action (mechanical, chemical, or both). This capsule can be totally or partially solubilized so that its contents (excipient and extraction particles) can come into contact with the sample or liquid that solubilized or weakened it, or dissociated in the case of deliberate mechanical solubilization / lysis.

[0072] It will be clearly understood that the capsules according to the invention are not intended for therapeutic, oral, or pharmaceutical use (as described in US2015 / 0147399). They are used for compartmentalizing extraction particles in a diagnostic context. The extraction particles contained within the capsules will be released when the capsule is ruptured and its outer shell is solubilized. These extraction particles will enable the capture of biomolecules contained in the sample to be tested, thereby allowing for a diagnosis.

[0073] Extraction particles are key elements for capturing biomolecules contained in the sample to be tested and are therefore essential to the test itself. They are crucial for efficient biomolecule capture and for the reliability and reproducibility of the test and diagnosis.

[0074] An extraction particle is defined as an identifiable, distinct, isolated physical structure that is insoluble in an aqueous medium, a mixture of organic and aqueous solvents, or an organic solvent, and whose size can be micrometric or nanometric. Preferably, the particle is nanometric and between 1 and 400 nm. This structure is capable of binding to biomolecules such as nucleic acids or proteins. This structure has a hydrophilic surface. Preferably, this structure is in the form of a particle or bead, for example, particles of magnetic compound(s) (maghemite, magnetite, etc.) coated with silica, as silica has a very strong affinity for nucleic acids under given conditions, or it is functionalized to capture proteins.These particles are generally magnetic due to their magnetic composition, meaning they are likely to react to a magnetic field with an orientation and / or displacement reaction dependent on the force and orientation. This force is exerted via the magnetic field and is generated by moving charges or magnets. This allows for the gathering, collection, and recovery of the extraction particles. The extraction particles of the invention preferably correspond to those described in patent application W02016108004A1.

[0075] The biomolecule extraction particles used preferentially in the invention are commercially available particles, such as Merck's SeraSil-Mag or MagPrep®, Thermofisher's DynaBeads, BioChain's PureSil, Macherey Nagel's NucleoMag, bioMérieux's NucliSENS® easyMAG® Magnetic Silica or MagSIL - particles or any other type of particle capable of binding to and capturing one or more biomolecules and preferably the nucleic acids contained in the sample to be tested.

[0076] In the capsule according to the invention, the volume of extraction particles is preferably about 50 µL. Since the particles must not dry out, this corresponds to the volume of extraction particles contained in the excipient. The total volume of particles and excipient is preferably 50 µL. The concentration of extraction particles varies from 5 mg / ml to 50 mg / ml. In a preferred embodiment of the invention, the capsule has an extraction particle concentration of 20 mg / ml. To facilitate the manufacture of the capsules according to the invention, their industrialization, and the industrialization of the test containers holding them, it is possible to use capsules with larger capacities ranging from 20 µL to 1 ml, preferably from 50 µL to 500 µL, and even more preferably from 50 µL to 150 µL. These figures are indicative and not limiting.Capsules with different volumes and / or concentrations of extraction particles can certainly be used. However, if the capsule has a capacity greater than 500 µL, it is important that its outer shell dissolves completely upon contact with water, aqueous buffer, lysis buffer, and / or the sample to avoid interfering with the subsequent biomolecule extraction process, particularly pipetting. A biomolecule is defined as a compound or chemical entity that may be a nucleic acid (DNA or RNA of any type, genomic DNA, complementary DNA, messenger RNA, complementary RNA, transfer RNA, mitochondrial RNA, chloroplast DNA, ribosomal RNA, plasmid DNA, viral, bacterial or yeast DNA or RNA, microRNA, snoRNA, siRNA, RNAi, in single- or double-stranded form (this list is not exhaustive or limiting) or a protein or any fragment of one of these entities.Preferably, the extraction particles of the present invention bind to one or more nucleic acids contained in the sample to be tested. The biomolecules may be free, circulating in the sample to be tested; they may also be constituents of a biological structure of the sample or contained within one of these structures.

[0077] In a preferred configuration of the invention, the biomolecules extracted by the extraction particles are nucleic acids.

[0078] By sample, we mean a sample having various origins such as samples of food, environmental, human, veterinary or cosmetic origin.

[0079] Examples of food samples include, but are not limited to, samples of dairy products (yogurt, cheese, etc.), meat, fish, eggs, fruit, vegetables, and beverages (milk, fruit juice, soda, etc.). Of course, these food samples can also come from sauces or more elaborate dishes, or from unprocessed or partially processed raw materials. A food sample can also be derived from animal feed, such as oilseed cakes or animal meal. All these samples, if not liquid, are pre-treated to achieve a liquid form.

[0080] As previously indicated, the sample may be of environmental origin and may consist, for example, of a surface sample, water sample, etc.

[0081] The sample may also consist of a biological sample, of human or animal origin, which may include samples of biological fluid (urine, whole blood or derivatives such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, bone marrow, etc.), stool (e.g., choleric diarrhea), samples from the nose, throat, skin, wounds, hair, organs, tissues or isolated cells, swab samples, bronchoalveolar lavage or sampling, and biopsies. This list is obviously not exhaustive.

[0082] Generally speaking, the term "sample" refers to a part or quantity, more specifically a small part or quantity, taken from one or more entities for the purpose of analysis. This sample may have undergone prior processing, involving, for example, mixing, dilution, or grinding, particularly if the starting entity is in solid form.

[0083] The sample analyzed is, in general, likely to – or suspected of – containing at least one biomolecule representative of the presence of microorganisms or of a disease to be detected, characterized or monitored. The sample can be tested directly after collection but it can also be tested after freezing, cold storage (for example at -20°C or 4°C), directly after a lysis step to obtain a lysate or after the lysate has been cold stored for preservation (for example at -20°C or 4°C or frozen).

[0084] Preferably, the sample, whatever it may be, is in liquid or semi-liquid form, that is to say, it may still contain solid, unliquefied materials or substances without this hindering the analysis that must be carried out on it.

[0085] The sample to be tested has a volume ranging from a few microlitres (about 5pl) to several millilitres (10 ml), preferably the sample is between 50pL and 1ml, more preferably between 100pl and 200pl.

[0086] Soluble in water, an aqueous buffer, a lysis buffer of the cells in the sample to be tested, and / or in the liquid sample to be tested, refers to a structure that dissolves or solubilizes at least partially in these media or environments, regardless of the temperature of the medium. Thus, under aqueous conditions (water, aqueous buffer, lysis buffer of the sample to be tested, or the sample itself), this structure or substance dissolves in the surrounding medium to form a homogeneous, uniform solution or mixture, a colloidal or non-coldoidal suspension, or an emulsion. Solubilization is not necessarily complete; it may be only partial, but it must allow the contents of the capsule to be released and mixed with the surrounding medium to form a solution, suspension, or emulsion.If solubilization is only partial, the unsolubilized portions or fragments do not interfere with the extraction process, preferably the extraction of biomolecules, implemented in the present invention. They do not interfere with any element used in this extraction process, either chemically or physically. Thus, if the capsule shell is not completely dissolved, the remaining suspended fragments do not physically hinder the capture of biomolecules by the extraction particles, nor the pipetting necessary during the biomolecule extraction process or other physical processes occurring during the extraction or detection of biomolecules, preferably nucleic acids. They also do not chemically interfere with the biomolecule extraction process.The solution or suspension created by dissolving or solubilizing the capsule shell and excipient in the medium containing the capsule does not interfere with or alter the structure of the biomolecules (nucleic acids and / or proteins) to be captured and extracted, nor does it alter the extraction particles that were contained in the capsule before its dissolution or solubilization. These particles retain all their capture and extraction capabilities in this new medium, which is the suspension or solution created following the partial or total dissolution or solubilization of the capsule shell and excipient.

[0087] It will be understood that by soluble, we mean not only that which can dissolve to form a homogeneous mixture once solubilized, but also that which is miscible without creating separation, a biphasic mixture, or a heterogeneous mixture. Given that the capsule necessarily contains extraction particles when it is not solubilized, it is understood that when we speak of a homogeneous mixture in its solubilized form, whether total or partial, we are referring to the mixture of the liquids outside the capsule, inside the capsule, and the solubilized structures that constituted the capsule in its solid form.

[0088] Solubility can thus be defined as the capacity of a substance (for example, the excipient) or material (film, capsule, etc.) to be broken down upon contact with a selected substance(s) within a given range. For example, Hansen parameters can be used for a chosen excipient or capsule substance, where soluble means that the Hansen parameters fall within the volume or solubility range for the material, as represented on a Hansen solubility chart (see "Hansen Solubility Parameter System," DuPont Nylon Intermediates and Specialties, publication W-400473, 12 / 2000). Alternatively, the capsule's capsule and excipient can be said to be dispersible in a given medium, such as water, an aqueous buffer, or the sample.They are also dispersible in a given medium through the aid of a physical means such as agitation, heating, shearing, the use of physical elements allowing the weakening of the envelope or a combination of two of these means.

[0089] The capsule shell and the excipient contained within the lumen or internal volume of the capsule are soluble in a defined external medium. This medium may be water, an aqueous buffer, a lysis buffer for the sample to be tested, or the sample to be tested itself in liquid form.

[0090] An aqueous buffer is an aqueous solution that resists pH changes despite the addition of small amounts of strong acid or strong base. This allows the pH of the solution to remain constant and thus prevents alterations to its properties. This aqueous buffer can be, for example, a solution of Tris HCl, sodium acetate, or suspended solids (SS), in concentrations between 10 and 100 mM, preferably 10 mM Tris HCl at pH 7 or a 10 mM sodium acetate solution at pH 5.5.

[0091] The aqueous buffer according to the present invention may contain a defined percentage of co-solvent such as, for example, an organic solvent, such as ethanol, isopropanol, or DMSO. Preferably, the co-solvent is in the proportions of 10% by weight (w / w), or more preferably 5% by weight (w / w), or even more preferably 2% by weight (w / w).

[0092] The aqueous buffer should preferably be used with a volume between 50 µL and 3 mL. This volume will be adjusted according to the sample size, the capsule size, and / or the instrument on which the test will be performed. This is well known to those skilled in the art, who will be able to adapt these choices.

[0093] A lysis buffer is a buffered solution capable of lysing the sample to be tested, the sample's cells, organelles, or membranes. Generally, a lysis buffer is an aqueous buffer that also performs lysis. It may contain, for example, a detergent and, if necessary, a chelating agent. The pH is typically maintained between 4 and 8, for example, between 6 and 8, using a suitable buffer such as Tris HCl, sodium acetate, or MES, possibly at concentrations between 10 and 100 mM.

[0094] The lysis buffer used in the present invention may comprise salts, for example NaCl, KCl, (NH3(ZiHSC)), preferably at a concentration between 0.05M and 0.5M.

[0095] The detergent can be non-ionic, anionic, cationic or zwitterionic and can be chosen from Tween, Thésit, Genapol X100, Ecosurf, Tritons, SDS, Brij and other commonly used detergents at concentrations between 0.05 and 20% by weight (w / w).

[0096] Optionally, the lysis buffer contains reagents for the inactivation of nucleases, and / or the removal of proteins, for example proteases such as proteinase K. Other enzymes can also be used such as lytic enzymes (hydrolase, zymolase ...) to digest the cell wall of yeasts and fungi.

[0097] In one specific embodiment, the lysis buffer does not include reagents for the removal of proteins, and in particular, proteases, specifically when the biomolecules to be captured are proteins.

[0098] In one specific embodiment, the lysis buffer also includes chaotropic agents. Chaotropic agents interfere with weak (non-covalent) intramolecular interactions, such as hydrogen bonds, van der Waals forces, and hydrophobic forces. Examples of chaotropic agents include urea, guanidine salts such as guanidinium chloride or thiocyanate, and lithium or sodium perchlorate. They are typically used in concentrations ranging from 1 to 6 M, particularly for GuSCN and GuHCl.

[0099] In another specific embodiment, the lysis buffer does not include chaotropic agents.

[0100] Chelating agents such as EDTA or similar compounds can also be added, for example between 5 and 50 mM and / or reducing compounds such as DTT (dithioerytreitol) or TCEP (tricarboxyethylphosphine) or beta-mercaptoethanol at concentrations of for example between 0.5 and 100 mM.

[0101] The lysis buffer may further comprise one or more organic solvents such as alcohols (ethanol, isopropanol, etc.) with a maximum of 30% by weight (w / w). In a preferred embodiment, however, the lysis buffer does not comprise any organic solvents.

[0102] The chemical characteristics of a sample must be considered when optimizing the lysis buffer. For example, the acidity of a sample (e.g., some soil samples) can damage nucleic acids and can therefore be neutralized to ensure a proper nucleic acid yield. The sample is exposed to the lysis buffer for a sufficient time to allow cell lysis without degrading the nucleic acids, for example, between 0 and 15 minutes.

[0103] The following lysis buffers are suitable for the present invention: ZYMO Research Lysis Buffer B - Reference: D2002-1-50, Qiagen RLT Plus Buffer - Reference: 1053393, MOBIO-Qiagen PowerLyzer® Lysis Buffer - Reference: 12855-50, Invitrogen eBioscience™ RBC IX Lysis Buffer - Reference: 00-4333-57.

[0104] The lysis buffer can have a volume between 100 µL and 3 mL, preferably between 200 µL and 2 mL. This will be adjusted according to the sample size, the capsule size, and / or the instrument on which the test will be performed. This is well known to those skilled in the art, who will be able to make these choices accordingly.

[0105] In one embodiment of the present invention, when a lysis buffer is used, it acts by lysing the cells and membranes of the sample, solubilizing the sample proteins while preventing their degradation and loss of biological activity, and regulating the pH and osmolarity of the lysate. Its purpose is to make the biomolecules to be analyzed accessible, where they will subsequently be captured by the extraction particles.

[0106] In one embodiment of the invention, the sample may be lysed beforehand, in which case the biomolecule extraction process according to the invention does not include a lysis step and no lysis buffer is used. In a preferred embodiment of the invention, the biomolecule extraction process from the sample to be tested includes a sample lysis step.

[0107] EXCIPIENT

[0108] An excipient is defined as an inactive substance that surrounds the extraction particles. In the present invention, the excipient is the medium in which the extraction particles are suspended but which has no interaction or interference with them.

[0109] The excipient is in liquid or viscous form. It is not in solid, powder, dry, dried, or dehydrated form. When the capsule shell is solubilized and the excipient comes into contact with the aqueous buffer, lysis buffer, and / or the sample used to solubilize the capsule shell, the excipient will be soluble in this new medium.Thus, the excipient is soluble in water, an aqueous buffer, a sample lysis buffer and / or the sample itself and is chosen from DMSO; diethyl formamide; acetonitrile; mono-alcohols, preferably ethanol, butanol, isobutanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, ethylene glycol, triethylene glycol; tri-alcohols, preferably glycerol; a mixture of at least two of these compounds; vegetable oils, preferably refined almond oil mixed or not with a mono-alcohol, preferably ethanol, a mixture of at least two of these compounds.

[0110] This excipient is chosen so as not to alter the performance of the extraction particles, nor to interfere with the capsule shell, weaken it, or dissolve it. It is therefore compatible with the performance of the extraction particles and also with the stability and integrity of the capsule shell. Once the capsule shell is dissolved or dissolved by the addition of the appropriate solution (water, aqueous buffer, sample lysis buffer, and / or the sample itself), the excipient comes into contact with the lysis buffer and / or the sample to be tested. It has no inhibitory effect or other negative interaction with these. Thus, the excipient can be said to be compatible with the aqueous buffer, the lysis buffer, and / or the sample to be tested.

[0111] In one embodiment of the invention, the excipient(s) contained in the capsule are monoalcohols selected from ethanol, isopropanol, terbutanol, dimethylpropanol, or a mixture of at least two of them. These compounds are compatible with the extraction particles, thus allowing their storage at room temperature within the capsule while retaining their properties and performance in capturing biomolecules, particularly nucleic acids. It is therefore advantageous to preserve the structure of the extraction particles, and thus their binding and / or biomolecule-capturing properties, as this extends the lifespan of the test (the container holding the capsule according to the invention and providing a reproducible and reliable result).

[0112] In a preferred embodiment of the invention, the capsule excipient is selected from DMSO, acetonitrile, glycerol and ethanol, refined almond oil with added alcohol, such as ethanol or glycerol, or a mixture of at least two of them.

[0113] According to one feature of the invention, the excipient concentration in the capsule is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. These percentages represent weight percentages (w / w). For example, the excipient concentration in the aqueous buffer represents 40% by weight of the excipient / aqueous buffer mixture.

[0114] The chosen excipient is preferably used at a concentration between 40% and 100%, except for ethylene glycol, which is preferably used at a concentration between 40% and 90%, and more preferably at a concentration between 40% and 80%, and vegetable oil, which is not used at 100% because it must remain perfectly soluble in water, an aqueous buffer, a lysis buffer for the sample, and / or the sample to be tested. In this case, refined or unrefined vegetable oil is used at a maximum concentration of 90% with a minimum of 10% alcohol, such as ethanol or glycerol. Among vegetable oils, refined almond oil is preferred. Even more preferably, refined almond oil at 90% with 10% ethanol is chosen.Naturally, different proportions of these compounds are possible while remaining within the limits indicated above to ensure the integrity of the capsule shell, the properties of the extraction particles, and the final solubility once the shell is fully or partially dissolved. These percentages represent weight percentages (w / w).

[0115] Preferably, the excipient is contained in water or an aqueous buffer in a proportion that does not allow the capsule shell to dissolve or solubilize. It is important to remember that it is the external addition of water, aqueous buffer, lysis buffer (aqueous), or the sample itself that will solubilize or dissolve the shell. Thus, the excipient, when not at a concentration of 100% by weight (w / w), is in a chosen aqueous buffer which may be, for example, the storage solution of the extraction particles (commercial storage buffer, original formulation), sodium acetate, MES (morpholino ethanesulfonic acid) in the form of potassium or sodium salt, a solvent which will allow the solubilization of the excipient but which will remain compatible with the integrity of the shell when the capsule is not solubilized and the performance of the particles.Preferably, this solvent is an alcohol, such as ethanol or glycerol.

[0116] In a particularly preferred embodiment of the invention, the excipient is at a concentration of at least 90% by weight (w / w). In a particularly preferred embodiment of the invention, the excipient is at a concentration of at least 90% by weight (w / w) in an aqueous buffer at a pH adapted to the stability and performance of the extraction particles, preferably in a MES buffer at pH 5.5 and even more preferably at a concentration of at least 95% by weight (w / w) in an aqueous buffer, or even 98% by weight (w / w).

[0117] In one embodiment of the invention, specific excipients are chosen depending on the target to be detected (biomolecules or subtypes of biomolecules, such as bacteria, viruses, DNA, or RNA). Thus, when RNA is to be detected, ethylene glycol, propanediol, and triethylene glycol are not preferred. Similarly, when viruses are to be detected, triethylene glycol is not the preferred choice.

[0118] In certain embodiments of the invention, envelope / excipient pairs can be chosen according to the targets to be detected. Thus, when the envelope is PVA, the excipient is preferably chosen from diethyl formamide; acetonitrile; mono-alcohols chosen from ethanol, butanol, isobutanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, triethylene glycol; tri-alcohols, preferably glycerol; a mixture of at least two of these compounds; a vegetable oil, preferably refined almond oil, mixed or not with a mono-alcohol, preferably ethanol.When the envelope is made of HPMC, the excipient is chosen from DMSO; diethyl formamide; acetonitrile; mono-alcohols, preferably ethanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, ethylene glycol, triethylene glycol; tri-alcohols, preferably glycerol; a mixture of at least two of these compounds; vegetable oil, preferably refined almond oil mixed with a mono-alcohol, preferably ethanol.When the capsule is made of gelatin, the excipient is chosen from DMSO; diethyl formamide; acetonitrile; mono-alcohols, preferably ethanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, ethylene glycol, triethylene glycol; tri-alcohols, preferably glycerol; a mixture of at least two of these compounds; vegetable oil, preferably refined almond oil mixed with a mono-alcohol, preferably ethanol.When the envelope is made of pullulan, the excipient is chosen from DMSO; diethyl formamide; acetonitrile; mono-alcohols, preferably ethanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, ethylene glycol, triethylene glycol; tri-alcohols, preferably glycerol; a mixture of at least two of these compounds; vegetable oil, preferably refined almond oil mixed with a mono-alcohol, preferably ethanol.

[0119] In a preferred embodiment of the invention, the capsule comprises a shell made of PVA, gelatin or pullulan and an excipient selected from glycerol or vegetable oil (preferably almond oil)

[0120] Preferably, the PVA / glycerol or gelatin / refined almond oil combination should be chosen.

[0121] Ideally, the excipient should not evaporate; therefore, it should be low-volatility, either alone or in mixtures, meaning it should have a boiling point above 75°C.

[0122] This also prevents the extraction particles contained in this excipient from drying out and thus from losing performance, i.e., their ability to capture biomolecules from the sample being tested. According to the invention, the particles are considered to be effective when they capture at least as many or more biomolecules, preferably nucleic acids, than under normal conditions without a capsule, which serve as a reference (see Example 4 with a comparison to a liquid reference). It should be clearly understood that the extraction particles in the capsule are not bound to one or more biomolecules (proteins or nucleic acids) or to an active agent when they are in the closed capsule. They only capture biomolecules when the capsule is in solubilized form, broken, and released into the external environment, no longer encapsulated or compartmentalized.Once in the external environment and in contact with the sample to be tested, the extraction particles can be considered an active agent that will act by capturing biomolecules from the sample. Under no circumstances are the extraction particles contained in the capsule bound to, carrying, or in the presence of an active ingredient, active agent, or molecule with therapeutic intent.

[0123] In a particularly preferred embodiment of the invention, the capsule consists solely of: a closed envelope soluble in an aqueous buffer, a cell lysis buffer of the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume, biomolecule extraction particles, preferably silica particles and more preferably magnetic silica particles, and an excipient in liquid or viscous form, soluble in an aqueous buffer, a cell lysis buffer of the sample to be tested and / or in the liquid sample to be tested, the biomolecule extraction particles and the excipient being contained in the internal volume delimited by the capsule envelope.

[0124] In a particularly preferred embodiment of the invention, the capsule consists solely of: a closed envelope soluble in an aqueous buffer, a lysis buffer for cells of the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume, magnetic silica particles having a hydrophilic surface, and an excipient in liquid or viscous form, soluble in an aqueous buffer, a lysis buffer for cells of the sample to be tested and / or in the liquid sample to be tested, the magnetic silica particles and the excipient being contained in the internal volume delimited by the capsule envelope.

[0125] For the purposes of this invention, "compatible" means suitable, non-interfering, coexisting without conflict, not disrupting, altering, or hindering the proper functioning of, not interacting with, or harming the structure or properties of, and inert with respect to. This means that the nature of the component mentioned does not disrupt the nature, structure, or, above all, function of the second component mentioned. For example, when it is stated that the excipient is compatible with the performance of the extraction particles, this means that the excipient is selected so that its nature does not disrupt the functions of the extraction particles and that, even in the presence of the excipient, the extraction particles will be able to capture biomolecules as effectively as they would without the excipient. One could say that the excipient is inert with respect to the extraction particles.The same applies when it is stated that the coating, once solubilized, is compatible with the performance of the extraction particles. This means that the chemical nature of the coating, when dissolved in the external medium—which will then consist of a liquid suspension comprising at least the sample to be tested, the compound(s) forming the coating, the compound(s) forming the excipient, and the extraction particles—will not disrupt or interfere with the structure, nature, and function of the extraction particles, which will then be able to capture the biomolecules of the sample to be tested as effectively as if the chemical compounds of the coating and the excipient were not present.

[0126] Similarly, when we say that the excipient is compatible with the stability of the capsule, this means that the nature of the excipient is selected so that it does not disrupt the stability of the capsule. In other words, the chemical nature of the excipient is chosen so that it does not react with the chemical nature of the compound(s) constituting the capsule, so that the capsule remains intact and stable, can maintain its function and act as a barrier against the external environment, containing both the excipient and the extracted particles.

[0127] Stability is defined as remaining in the same state and having its structure and properties unaltered, unweakened, or unmodified. Thus, when it is stated that the excipient is compatible with the stability of the capsule shell according to the invention, it is indicated that the excipient does not weaken, modify, or alter the structure of the shell, which remains intact in the presence of the excipient it contains when in its unsolubilized form. The properties of the shell in its solid, unsolubilized form are not altered by the excipient, nor are the extraction particles contained within the internal volume defined by the closed shell. The shell therefore acts as a barrier to the external environment and thus protects the excipient and the extraction particles from said environment.When we say that an excipient is compatible with the properties of the extraction particles, we mean that the excipient does not harm or alter the structure of the extraction particles, nor their ability to capture biomolecules, and in particular nucleic acids. Therefore, the excipient does not negatively interact with the physicochemical properties of the extraction particles, and their extraction yield remains unchanged.

[0128] Furthermore, when it is said that the envelope is compatible with the properties of the extraction particles, it is indicated that the chemical structure of the envelope does not interfere with the chemical or physical structure of the extraction particles, nor with their ability to capture biomolecules, and this is true whether the envelope is in its solid, unsolubilized form or in its solubilized form in the external medium (water, aqueous buffer, lysis buffer and / or sample) with the addition of the excipient that it released when solubilizing to form a suspension or a solution.

[0129] Sample lysis can be mechanical, chemical, or a combination of both. Various techniques can be used for lysis, for example, grinding with solid elements, high-pressure homogenization, sonication, ultrasonication, osmotic shock, freeze / thaw lysis, and the use of chemical compounds such as detergents and lytic enzymes (lysozyme, proteinase K, etc.). More specifically, lysis by grinding is a technique that destroys the cell wall using shear stress created by solid elements, preferably particles or beads. These particles can be made of glass, silica, ceramic, zirconium, or metal, and may or may not be magnetic. They are agitated in a sealed container in the presence of the cells to be ground. Agitation can be internal or external to the container and generated by blades, magnetization, or a vortex, for example.

[0130] Sample lysis can be chemical, achieved using a lysis buffer whose components break down the chemical structures of cell membranes to release their contents (DNA, RNA, proteins, organelles) and solubilize proteins without degrading them. Chemical lysis is carried out using ionic detergents, such as Triton, which preserve protein activity, or non-ionic detergents, such as SDS, which disrupt and break down membranes and proteins. Chemical lysis can also be performed with enzymes such as lysozyme and proteinase K. A combination of mechanical and chemical lysis can be considered, resulting in a more complete and rapid lysis. In the present invention, a combination of both types of lysis is preferred.

[0131] Extraction refers to a technique for isolating biomolecules from any sample, for example, the isolation of DNA or RNA from eukaryotic, prokaryotic, human, animal cells, microorganisms, or tissue. Thus, extraction as defined in the invention includes the lysis of the sample and the capsule of the invention, and the purification of the biomolecules. Purification itself comprises the adsorption or capture, washing, and elution of the biomolecules and / or microorganisms. Capture consists of adsorbing the biomolecules and / or microorganisms onto the extraction particles, and elution consists of their desorption or release from the extraction particles.

[0132] The invention also covers a container or extraction device containing at least one capsule according to the invention.

[0133] The test container may also be called a test or extraction device or container. It may be an Eppendorf® type tube, a jar, a bottle, a cartridge or strip (Vidas® type strip), a FilmArray® type flexible pouch, with or without a microfluidic circuit, a multi-well plate whose wells are all of the same size or of different sizes, or any type of container suitable for holding a capsule according to the invention and in which a molecular biology test (extraction, amplification and detection), and in particular an extraction, can be carried out.

[0134] The volume of the container is adapted to the volumes chosen to carry out the test and in particular the volume of the sample to be tested and the volume of the capsule according to the invention.

[0135] The container is preferably made of plastic, but it can also be made of metal, glass, or any inert material or material that does not interact with the sample and the substances used for the test (buffer, enzyme, extraction particles, capsule shell, capsule excipient, etc.). The container can be closed or open. In a preferred embodiment of the invention, the container is closed. Closure can be achieved with a cap, a screw, a lid, or a foil, which can be aluminum foil, a plastic film, or a multilayer composite. Generally, the lid is thin, allowing for a tight seal by heat sealing or adhesive sealing.

[0136] The container of the invention contains the capsule when it is a single-well container, but when it has multiple wells, it can contain reagents that enable the various stages of the extraction process according to the invention or the various stages of the detection process according to the invention. Thus, the container can include washing solutions, an amplification buffer comprising, among other things, nucleotides and a polymerase enzyme, a detection reagent, and / or a lysis buffer.

[0137] MANUFACTURING PROCESS

[0138] The invention also relates to a method for manufacturing a capsule according to the invention comprising the following steps:

[0139] Prepare a suspension of biomolecule extraction particles in an excipient or mixture of excipients,

[0140] Prepare a capsule envelope or use a pre-formed capsule envelope,

[0141] Fill the internal volume delimited by the envelope with the suspension of particles extracted from the biomolecules in the excipient or mixture of excipients,

[0142] Seal the envelope so that it is airtight.

[0143] The invention also relates to a method for manufacturing a capsule according to the invention, which comprises the following steps:

[0144] Prepare a suspension of extraction particles at a given concentration in the desired excipient or mixture of excipient(s) compatible with the nature of the shell that will be used to form the capsule,

[0145] Prepare a capsule envelope or use a pre-formed capsule envelope,

[0146] Fill the envelope with the suspension of extraction particles using suitable equipment, and close the envelope so that it is airtight.

[0147] To prepare a suspension of extraction particles suitable for insertion into a capsule according to the invention, and more specifically into the internal volume defined by the capsule's outer shell, extraction particles suspended in their storage buffer or in the buffer in which they are sold must be taken. The quantity of extraction particles depends on the chosen capsule size. Generally, a quantity ranging from 20 µL to 1 mL of this suspension is taken.

[0148] If the particles do not need to be magnetized, the supernatant (storage buffer) of the suspension will be removed by pipetting. It can also be removed after decantation, appropriate centrifugation, or filtration. The tools and settings are well known to those skilled in the art and will be adapted according to the type of extraction particle chosen, the volume, and the specific requirements.

[0149] If the extraction particle suspension is not magnetized and requires magnetization, it is magnetized using appropriate equipment, for example, on a magnetic rack equipped with neodymium / boron magnets or an electromagnet. This magnetization is carried out for a suitable duration, ranging from a few seconds (approximately 10 seconds) to a few minutes (approximately 5 minutes). Once the extraction particles are magnetized, they are immobilized using a magnetic field, and the supernatant is removed using the same techniques as for non-magnetized particles. Preferably, the supernatant is removed by pipetting or pumping.

[0150] The supernatant is replaced by a suitable excipient solution, preferably with a volume equivalent to the volume of supernatant removed. Naturally, the volume of excipient added will be adjusted according to the requirements and the desired final concentration of extracted particles. In a preferred embodiment of the invention, the final suspension of extracted particles is 20 mg / ml in the desired excipient. The excipient concentration can also vary, as detailed above in the section dedicated to the excipient. It is understood that the supernatant can be replaced by a mixture of excipients suitable for the capsule according to the invention and also defined in the section dedicated to the excipient.

[0151] In a preferred embodiment of the invention, the replacement of the supernatant (storage buffer) is carried out repeatedly, preferably twice, in order to ensure that there is no longer, or only very small, amount of storage buffer with the extraction particles and only and almost only the excipient or excipients as defined above.

[0152] The preparation of the capsule shell is carried out in a conventional manner for a person skilled in the art. The ingredient or component of the capsule shell according to the invention must be selected. This is detailed above in the paragraphs dedicated to the shell. Once the main constituent of the shell has been selected, water, preferably hot (at least 40°C), is added and mixed to obtain a homogeneous liquid or viscous solution. Naturally, colorants, preservatives, or any other necessary components suitable for the capsule according to the invention can be added to this homogeneous solution.

[0153] The homogeneous solution can then be poured into molds shaped like the capsule as defined above. Multiple molds may be required when the capsule is in several parts, as is often the case with a capsule-shaped device, which typically has a male and a female part. Alternatively, the homogeneous solution is poured onto a plate and spread to form a film. This technique is used when the capsule is in the form of a sachet, bag, or carton. Other techniques adapted to shape the homogeneous solution and form the capsule shell can be used. The mold, plate, or other chosen material is then cooled to solidify the shell. Cooling can be done in open air or with fans, in a refrigerator, a cold room, or any other suitable system.

[0154] In one embodiment of the present invention, it is possible to choose a pre-formed capsule shell. These shells may have been previously manufactured and stored, or they may also be commercially available. For example, suppliers such as LGA (La Seyne-sur-Mer, France) or Aromazone (France) may be available.

[0155] The filling of the capsule shell, and more specifically the internal volume defined by the shell, can be done in several ways: manually, with a syringe, a needle, a spatula, a spoon, or any other suitable utensil or instrument for precise filling and dosing. This can be done manually, semi-automatically, or fully automatically. A person skilled in the art will know how to choose the appropriate equipment based on the shape and nature of the shell and the excipient(s). When the capsule is in several parts, only one part is filled first, and then the capsule is closed with the second part. When the extraction particle suspension is liquid, only one part can be filled. However, when the extraction particle suspension is viscous, it is possible to at least partially fill both parts and then assemble them.It will be understood that "filling the envelope" means filling the internal volume defined by the envelope.

[0156] When the capsule shell is already closed, it is possible to make a small incision or pierce the shell with a syringe or any suitable utensil to inject the particle suspension.

[0157] The capsule shell is then closed either manually or automatically. Depending on the final shape of the capsule, closure can be achieved by screwing two parts together (male and female), by interlocking (male and female), by hot or cold assembly, by sealing, by gluing, by heat sealing between 100°C and 200°C, preferably between 120°C and 160°C, by adhesive, by pressure, by applying a small part compatible with the functions of the capsule and the nature of the shell and filling a hole or incision, for example when filling has been done by piercing an already closed shell.

[0158] In one embodiment, to ensure the capsule's airtightness, a seal can be added at the junction between the two capsule parts or between two sections of the envelope, where there is an overlap or fold. This seal can be made of plastic, silicone, rubber, or any other material compatible with the capsule's functions and the nature of the envelope, and which does not interfere with the extraction or detection process according to the invention. This seal can be applied in liquid form and then hardened and dried, or it can be already solid and applied by gluing, heating, or sealing. Airtightness can also be achieved by welding with a suitable solvent or a gelatin strip.

[0159] Of course, a skilled professional will know how to choose the appropriate techniques and components depending on the capsule to be made. TEST CONTAINER FILLING PROCESS

[0160] The invention also relates to a method of filling an extraction container with a capsule according to the invention.

[0161] It is clear that the container is adapted to the size of the capsule. This filling process consists of fitting a container with a capsule according to the invention. When the container has several wells, only one of the wells contains a capsule. The capsule can be added to the container well in advance. This is the case when the container is ready to use and the operator or the automated system performing the test only has to add the sample to be tested. As previously mentioned, the capsule included in the container will prevent the extraction particles contained within the capsule from dispersing onto the container walls during transport or handling. The distribution of the capsules into the container is done automatically or manually. The automated method will be preferred because it saves time, is more reproducible, and does not cause musculoskeletal disorders for the operator.When the container is filled, it is capped or resealed to facilitate transport.

[0162] In one embodiment, the test container or extraction container is filled by the operator or the automated test system / instrument directly at the time of testing, i.e., as soon as the sample to be tested is available. This method can be used when the container has no lid or seal and transporting the container already containing the capsule would have been complicated. This filling method has the advantage of preventing the dissemination of extraction particles onto the container walls when the sample or an aqueous or lysis buffer is added (splashing), and also of preventing contamination of the automated system or the operator's work surface.

[0163] EXTRACTION PROCESS

[0164] The invention also relates to a method for extracting biomolecules (nucleic acids and / or proteins) from a sample to be tested.

[0165] This method for extracting biomolecules from the sample to be tested comprises: a) A step of contacting the lysed or unlysed sample with the capsule according to the invention, the solubilized contents of the capsule or only the extraction particles previously contained in the solubilized capsule according to the invention, b) A step of lysing the sample before and / or after contacting the unlysed sample with the capsule, the solubilized contents of the capsule or the extraction particles previously contained in the capsule, c) A step of capturing the biomolecules from the sample to be tested by the extraction particles, d) Optionally, a step of washing the biomolecules captured by the extraction particles, e) Optionally, a step of eluting the biomolecules captured by the extraction particles.

[0166] The sample to be tested can be collected a few hours or days before the test or immediately beforehand. To be tested, it must be added to the container in which the test / diagnosis will be performed. As previously mentioned, the container can be a cartridge, a plate, a tube, a strip, etc. When the container has several wells or when there are several containers, the sample is added to a well containing a capsule according to the invention. The extraction process of the invention can therefore include a step of adding the capsule according to the invention to a container, as well as a step of adding the sample to be tested or a pre-lysed sample to be tested to the test container.

[0167] In one embodiment, the sample is added to the container first, and the capsule is then added to the sample already present in the container. In both cases, the sample will solubilize the capsule, but it is also possible to subsequently add water, an aqueous buffer, and / or a lysis buffer to this mixture, which will also contribute to the solubilization of the capsule.

[0168] It is also possible that the capsule is present or added to a well of the container or to a given container, and that the sample is added to another well of the container or to another container not containing a capsule. In this case, only after the capsule has been solubilized with water or a suitable buffer are the extraction particles released into the well where the unsolubilized capsule was located and then transported to the well or container containing the sample. In this case, if the sample had not been lysed before being added to the container or before contact with the extraction particles, it is necessary to add a sample lysis step. It is also possible that the sample, already lysed or having been lysed in a well of the container or in a given container, is moved to the well containing the solubilized capsule.It should therefore be understood that the sample and the capsule may be in the same well of a container or in separate wells or containers and then be found together. If there has been no sample lysis step, one should be performed before the extraction step.

[0169] In one embodiment of the invention, the sample may have been previously lysed and may or may not have been frozen. In this case, there is no additional lysis step, and the capsule is solubilized either by the sample itself or by water, an aqueous buffer, or a lysis buffer in a well or container separate from the one containing the sample.

[0170] The solubilization of the capsule occurs either chemically by water, an aqueous buffer or the sample itself, or by a mechanical action (which can also be called mechanical lysis) which weakens and breaks the capsule's outer layer, or by a chemical and mechanical action.

[0171] In a preferred embodiment of the invention, the capsule is solubilized in a container or well separate from the container or well containing the sample, and the extracted particles are transported to the container or well containing the lysed sample.

[0172] Once the capsule is solubilized, its contents (excipient and extraction particles) are released and the biomolecule capture step can therefore be carried out by the extraction particles.

[0173] After the capture step, one or more washing steps can be carried out in the presence of a wash buffer, allowing the removal of contaminants without detaching the biomolecules (nucleic acids and / or proteins) from the extracted particles. The washing step may, for example, include a washing step in water or a suitable wash buffer, such as a low-salt solution possibly containing an alcohol, and / or a washing step in the presence of an alcohol to remove the salts. In a preferred embodiment of the invention, at least one washing step is performed. The wash buffer may, for example, comprise Tris HCl or sodium acetate or MES, possibly in concentrations between 10 and 100 mM. It may be an ethanol-based washing solution, for example, Ethanol: 70-80%, Tris-HCl: 10 mM (pH 7.5-8).0) and EDTA: 1 mM, or a salt-based wash solution, for example NaCl: 100-200 mM, Tris-HCI: 10 mM (pH 7.5-8.0) and EDTA: 1 mM, or a guanidine-based wash solution, for example Guanidine hydrochloride: 50-100 mM, Tris-HCI: 10 mM (pH 7.5-8.0) and EDTA: 1 mM.

[0174] If necessary, a subsequent step can be performed to elut the biomolecules (nucleic acids and / or proteins). The elution step consists of releasing the biomolecules retained on the extraction particles. A basic buffer, for example, a pH between 8 and 10, and a low ionic strength, such as Tris or borate buffer, are generally used. This is preferably carried out at a temperature between 50°C and 90°C. When the biomolecules are nucleic acids, elution is preferably performed with commercial products such as those in the NucliSENS® easyMAG® extraction range (bioMérieux, France). These conditions are standard and ensure the integrity of the nucleic acids.

[0175] In a preferred embodiment of the invention, the biomolecule extraction process includes an elution step and even more preferably it includes at least one washing step and one elution step.

[0176] The invention covers a method for extracting biomolecules contained in the sample to be tested, wherein the solubilization of the capsules or solubilization step of the capsules is carried out by adding water, an aqueous buffer, a lysis buffer of the sample, by the sample, by shearing, by agitation, by abrasion, by heating, by ceramic beads, by an element configured to at least weaken, crack or break the capsule shell, or any object weakening the capsule shell or by a combination of at least two of them.This solubilization of the capsules in this extraction process as described above can be carried out before step a), during step a) or during step b), with steps a) and b) defined as follows: a) a step of bringing the lysed or unlysed sample into contact with the capsule according to the invention, the contents of the solubilized capsule or only the extraction particles previously contained in the solubilized capsule according to the invention, b) a step of lysis of the sample before and / or after bringing the unlysed sample into contact with the capsule, the contents of the solubilized capsule or the extraction particles previously contained in the capsule.

[0177] If, during step a), the lysed or unlysed sample is brought into contact with the contents of the solubilized capsule or the extraction particles previously contained in the solubilized capsule, then the capsule was solubilized before step a). Similarly, if, during step b), the contents of the solubilized capsule or the extraction particles previously contained in the solubilized capsule are used, then the capsule was solubilized during step a) or before step a).

[0178] If in step a) an entire capsule that has not yet been dissolved is used, it will be dissolved in step a) or b). If in step b) an entire capsule that has not yet been dissolved is used, it will be dissolved in step b).

[0179] The methods used for the mechanical lysis of the sample can also be used for solubilizing the capsule, meaning that this involves physically rupturing the capsule's outer layer. These methods are known to those skilled in the art and are commonly used in molecular biology, particularly for lysing cell membranes and weakening biological structures. When a mechanical action is used to rupture or weaken the capsule's outer layer, this can also be referred to as solubilizing the capsule. This solubilization is not necessarily complete, but it will be initiated by the mechanical action and continued by contact with the sample. In a preferred embodiment of the invention, solubilizing the capsule is achieved through a combined or successive mechanical and chemical action.

[0180] In a particularly preferred embodiment of the invention, in the extraction process, the extracted biomolecules are nucleic acids.

[0181] Nucleic acids thus purified by the process described above (with or without washing and / or elution step) are usable in particular for in vitro amplification and detection applications.

[0182] DETECTION METHOD

[0183] Indeed, the present invention also covers a detection method which corresponds to the extraction method described above and further comprises:

[0184] - Optionally, a nucleic acid amplification step when the biomolecules extracted at the end of the extraction process are nucleic acids, preferably the amplification is carried out by PCR, A biomolecule detection step.

[0185] Alternatively, the detection or amplification step (when the biomolecules are nucleic acids) is performed directly after the lysis, capture, and optionally washing and elution steps. Preferably, the process includes a washing and elution step as well as an amplification and detection step. This amplification and detection can be performed directly on the extraction particles that have captured the biomolecules without a prior elution step. This is even more preferably done on a magnetic or non-magnetic extraction particle.

[0186] Amplification or amplification reaction refers to any nucleic acid amplification technique well known to those skilled in the art.

[0187] The amplification step is generally performed because the quantity of nucleic acids to be detected and / or quantified is very small, and an amplification phase is necessary to detect and / or quantify them in order to provide an accurate diagnosis. Without an amplification phase, and if the initial quantity of nucleic acids in the sample is low, it is entirely possible to obtain erroneous results concluding that a target nucleic acid is absent from a sample when, in reality, it is present but in such small quantities that the techniques used cannot detect it.

[0188] In one specific embodiment, the amplification of the detection method according to the invention is carried out using a DNA polymerase, such as Taq polymerase, Pfu polymerase, T7 polymerase, the Klenow fragment of E. coli DNA polymerase and / or a reverse transcriptase, or any other polymerase.

[0189] In another embodiment, the amplification of the detection method is a polymerase chain reaction (PCR), well known to those skilled in the art. The PCR protocol comprises, for example, 20 to 40 cycles, each cycle including at least (i) a denaturation phase of the DNA to be amplified at a temperature generally between 90°C and 95°C, (ii) a primer hybridization phase with the DNA to be amplified at a temperature generally between 55°C and 65°C, and (iii) an extension phase at a temperature generally between 68°C and 75°C. Variations of nucleic acid amplification methods by PCR can also be implemented. These include, in particular, nested PCR, quantitative PCR (qPCR), semi-quantitative or real-time PCR, error-prone PCR, and reverse transcription PCR (RT-PCR).It is also possible to implement other amplification techniques such as LAMP, NASBA, TMA, RPA, LCR, RCR, 3SR, RCA, SDA or any nucleic acid amplification techniques known to a person skilled in the art.

[0190] The biomolecule detection step is performed using appropriate detection methods. The standard detection methods available are all those widely known to those skilled in the art. Examples include, but are not limited to, radioactive labeling; non-radioactive labeling (colorimetry, fluorescence, chemiluminescence); molecular hybridization; Southern blotting; Northern blotting; dot blotting; and in situ hybridization. Preferably, detection is performed using a detection probe or a fluorogenic intercalating compound whose fluorescence is enhanced in the presence of DNA.

[0191] A detection probe, or simply a probe, is a nucleic acid sequence consisting of four different nucleotides chosen from the adenine, thymine, guanine, uracil, and cytosine groups. This sequence is capable of specifically hybridizing to an amplicon and includes at least one marker. The probe may be a round probe (see W010001074), a molecular beacon, a TaqMan® probe, or a FRET probe. The latter three types are well known to those skilled in the art. These probes may be composed entirely or partially of modified nucleotides. Each probe includes a marker and possibly a quencher.Among these probes, preferred methods include those that emit fluorescence upon hybridization to the complementary sequence, such as those described by Tyagi & Kramer (Nature Biotech, 1996, 14:303-308), commonly known as "molecular beacons," or commercial kits such as the R-gene® range from Argène (bioMérieux, Verniolle, France). Detection can be performed in real time or at the end of the reaction.

[0192] When the biomolecules to be detected are proteins, techniques widely known to those skilled in the art can be used for protein detection and quantification. These include, for example, Western blotting, which also uses an antibody, and colorimetry, chemiluminescence, or fluorescence; ELISA (Enzyme-Linked Immunosorbent Assay); UV spectrophotometry; colorimetric methods (BCA (Bicinchoninic Acid) or Bradford); flow cytometry; and fluorimetry, for example, with GFP (Green Fluorescent Protein).

[0193] The tests carried out for detection with or without amplification of target nucleic acids as well as the tests for detection of proteins with the capsules according to the invention give excellent results.

[0194] EXAMPLES

[0195] Example 1 - Generic extraction protocol and PCR analysis of nucleic acids from a sample

[0196] In order to evaluate the compatibility of the different excipients and encapsulants with maintaining the extraction performance of magnetic silica particles, the following protocol is implemented:

[0197] A fecal swab sample is taken from feces. It is placed in a liquid transport medium (Copan Fecal swab (reference 470CE.A)) in order to release the biological matrix by vortexing.

[0198] We have mother strains of model microorganisms that can be used in mixtures: Salmonella enterica (SLM), Yersinia enterocolitica (YER) from ATCC strains (ATCC 9263 for SLM and ATCC 55075 for YER), Adenovirus 40 (ADV) and Rotavirus (ROTA) from an internal source (bioMérieux, Verniolle, France), BK Virus (BKV) ordered from Zeptometrix in glycerolized stock (SKU: 0810065CF, NY, USA).

[0199] Live cells of some of the above microorganisms are added to the fecal sample to obtain respective concentrations of 8x0e3cp / mL in SLM, 2.6x0e3cp / mL in YER, 6.7x0e2cp / mL in ADV, 3x0e3cp / mL in BKV or 2.17 cp / mL in ROTA.

[0200] A 200 µL volume of the sample as prepared above is then processed on an automated eMAG® nucleic acid extraction platform (BioMérieux, Marcy l'Etoile, France) equipped with NucliSENS® extraction reagents (lysis buffer, washing buffers and elution buffer, magnetic silica beads, etc.) following the supplier's protocol (BioMérieux 50). In short:

[0201] The 200pl of the biological fecal sample as prepared above are added to 2ml of NucliSENS® eMAG® Lysis Buffer to which 50pl of the commercial magnetic particle suspension at 20 mg / ml (MagSIL NucliSENS® easyMAG® Magnetic Silica ref 280133, bioMérieux, Marcy l'Etoile, France) is added as a reference.

[0202] The preparation is treated successively by the washing and elution buffers while magnetization cycles are carried out by the automaton.

[0203] The eluate obtained after the extraction described above, containing the nucleic acids of the different microorganisms, is then mixed with various PCR premixes containing the necessary components to amplify the corresponding nucleic acids. These premixes are derived from the BK R-GENE® 69-013B kit for the detection of BK viruses and from ad hoc PCR formulations, each containing 0.4 pM of primers, 0.2 pM of TaqMan® probe, and 0.4 U / pL of Taq polymerase (and 1 U / pL for Rota (Reverse Transcriptase)) for the detection of SLM, ADV, ROTA, and YER, respectively. Each BK virus amplification was performed according to the recommendations of the BK R-GENE® kit.For the other microorganisms, the following PCR conditions were used: 7.5 µl of premix mixed with 7.5 µl of eluate, with a reverse transcription step (when necessary) for 5 minutes at 55°C per cycle, a Taq polymerase activation step for 2 minutes at 95°C per cycle, a denaturation step for 3 seconds at 95°C, and a hybridization step for 35 seconds at 60°C for 45 cycles (denaturation and hybridization). PCR detection was performed using TaqMan® technology on a CFX 96 thermocycler (BioRad, Hercules, USA) according to the manufacturer's recommended protocol to collect Ct (Cycle Threshold) values ​​characteristic of magnetic silica extraction performance.These Ct values ​​will allow us to evaluate the performance of the extraction particles under different conditions, that is, under the different conditions of the magnetic extraction particles, but also under the choice of capsule components (shell and excipient). The results are shown below.

[0204] Example 2 - Comparison between nucleic acid extraction with dried and undried extraction particles.

[0205] A fecal sample is extracted and amplified according to the protocol given in Example 1 in the presence, on one side, of commercial particles (MagSIL NucliSENS® easyMAG® Magnetic Silica ref 280133, bioMérieux, Marcy l'Etoile, France - noted Magnetic Particles REF LIQUID in the table) in liquid suspension in the aqueous storage buffer in which they are sold and ready to use and, on the other side, with the same quantity of the same commercial particles but previously dried by evaporation in open air and at room temperature (noted DRIED Magnetic Particles in the table).

[0206] Table 1 below shows the observed performance of SLM, YER, and ADV extractions under each condition (liquid-based extraction particles and dried extraction particles). Amplification control showed no difference; there was no PCR inhibition for any of the amplifications (not shown). The Ct (Cycle Threshold) corresponds to the number of cycles required for the fluorescence generated by the PCR product amplification to exceed a detectable threshold level. The Ct value is inversely proportional to the initial amount of the target in the sample: the lower the Ct value, the higher the initial amount of the target.

[0207] When the extraction particles are dried, a Ct delay is observed in all three PCR assays (Mean Ct SLM, YER, ADV) compared to the liquid reference. Since there is no PCR inhibition, the delay is necessarily due to the initial quantity of nucleic acids and therefore to the extraction performance of the particles. This Ct delay (calculated as the difference between the Ct of the liquid reference and the Ct of the dried particles, or Delta Ct) is greater than 1 and indicates that the extraction was less efficient with the dried particles (almost 10 times less DNA extracted for YER, for example).

[0208] It is therefore clear that the drying of magnetic silica extraction particles impacts the nucleic acid extraction performance of these particles. Consequently, the extraction particles must not be dry or dried out if good sensitivity and speed of the PCR test are to be maintained. A solution must therefore be found to prevent these particles from drying out.

[0209] [Table 1] - Comparison between nucleic acid extraction and amplification with dried and undried extraction particles

[0210] Example 3 - Preparation of extraction particles and formation of a capsule according to the invention

[0211] A 1 ml suspension of magnetic particles (MagSIL NucliSENS® easyMAG® Magnetic Silica ref 280133, bioMérieux, Marcy l'Etoile, France) at 20 mg / ml is magnetized for 2 minutes on a magnetic rack (Dynal, Oslo, Norway), and the supernatant is then removed by pipetting. It is replaced with an equivalent volume of a solution of one of the excipients chosen for the invention (see Example 4) at various concentrations in 50 mM MES pH 5.5 or in ethanol. These steps are repeated once to remove and replace the aqueous solution of the initial magnetic particle suspension. The final extraction particle suspension is at 20 mg / ml in the desired excipient.

[0212] The suspension is introduced into a capsule (A), a water-soluble polymer sachet (B) or an ellipsoidal capsule (C). (A) and (C) were already formed and commercially available.

[0213] A) Capsule according to the invention in the form of a hard pullulan capsule (A in Fig. 4). For this purpose, the suspension of magnetic particles in the excipient as described in the first part of Example 3 (here glycerol at 90% w / w in MES pH 5.5 at 50 mM) is introduced using an automatic pipette into the male part of one half of a type 4 gelatin capsule (LGA, La Seyne-sur-Mer, France). The male part of the capsule is then closed with the female part.

[0214] B) Capsule according to the invention in the form of a sachet made of polyvinyl alcohol (B in Fig. 4) Two sheets of polyvinyl (PVA) film HF / F7 24mg / m 2CM-100 (ECOMAVI, Italy) are heat-sealed at approximately 140°C for 10 seconds to form a tubular conduit closed at one end. A hollow needle is used to fill this conduit with the suspension of magnetic extraction particles in the desired excipient as described in the first part of Example 3 (here, 95% w / w glycerol in 50 mM MES buffer pH 5.5). The conduit is closed at the other end by the same heat-sealing process at 140°C for 10 seconds. Once the conduit is closed at both ends, a further heat-sealing at 140°C creates solid sections along the conduit, which will form capsules of the invention in the form of sachets. These sachets can then be cut and dispensed into the test container or plastic consumable. The size of the sachets is adapted to the quantity of magnetic particles to be delivered (generally between 0.5 and 2 mg).In this example, a suspension of 50pl at 20mg / ml was used, i.e. 1mg of extraction particles.

[0215] C) Soft ellipsoidal capsule according to the invention (C in Fig. 4)

[0216] A soft, ellipsoidal capsule made primarily of gelatin is filled with the extraction particle suspension defined in the first part of Example 3 (here in the excipient refined almond oil, 98% w / w in ethanol) using a needle and syringe. With the capsule shell closed and airtight externally, the needle is inserted into the shell to access the empty space inside, or internal volume. Using the syringe, this internal volume is filled with the aforementioned extraction particle suspension to a volume of 50 µL. The capsule is sealed by a small piece of gelatin applied to the opening created by the needle and sealed with ethanol.

[0217] It is observed that the manufacturing process of the capsules gives stable capsules for several months (a minimum of 3 months) in which the excipient does not affect the stability and integrity of the shell which does not dissolve without external action and in which the extraction particles are functional.

[0218] Example 4 - Choice of capsule excipients and performance of extraction particles

[0219] A fecal sample is extracted and amplified according to the protocol given in Example 1 in the presence, on one side, of commercial particles (MagSIL NucliSENS® easyMAG® Magnetic Silica ref 280133, bioMérieux, Marcy l'Etoile, France - noted as Reference magnetic particles of the day of the experiment 1 to 5 - REF LIQUID 1 to 5 in Table 2) in liquid suspension in the aqueous buffer for storing the extraction particles and, on the other side, with the same quantity of the same particles taken up in a given excipient (see first column of Table 2) at different concentrations (see second column of Table 2) in the MES pH 5.5 to 50 mM.

[0220] The amplification control showed no difference; there was no inhibition of PCR for each of the amplifications.

[0221] Thus, we were able to evaluate the effect of excipients at different concentrations in MES pH 5.5 to 50 mM on the performance of the extraction particles for nucleic acid extraction and the efficiency of PCR. A comparison was made for each series of excipients against a reference magnetic silica from the day of the experiment (liquid REF 1 to 5).

[0222] Table 2 allows us to calculate, as shown in Table 3, the difference in Ct between manipulations performed with the reference magnetic silica and the magnetic silica used in a given excipient. A Ct difference of + / - 1 is considered to have no impact on nucleic acid extraction from the sample, nor on PCR, while a Ct difference > 1 is considered to have a significant impact. Conversely, a Ct difference < 1 is considered to indicate a significant improvement in nucleic acid extraction.

[0223] [Table 2] - Extraction and amplification performance with different excipients and concentrations.

[0224]

[0225] Table 3 demonstrates that the selected and tested excipients do not cause a decrease in nucleic acid extraction performance (Ct difference + / - 1 for all excipients). In the case of glycerol, an improvement in BKV extraction performance is even observed (Ct difference greater than 1). The described excipients are therefore good candidates for reabsorbing the extraction particles (magnetic silica) in suspension, as they have little to no impact on nucleic acid extraction. They are thus compatible with the performance of the extraction particles.

[0226] [Table 3] - Difference between the Ct of the liquid reference and the Ct obtained with the excipient.

[0227]

[0228] Example 5 - Choice of capsule shell components and extraction particle performance

[0229] Materials used to construct the capsule shell (capsule, sachet, etc.) were selected and evaluated to verify their impact on the extraction and amplification of nucleic acids contained in the sample to be tested. The same sample and operating conditions (extraction and amplification) as in Example 1 were used.

[0230] A polyvinyl alcohol film (PVA, ECOMAVI, Italy), a gelatin capsule (GELATIN, LGA, La Seyne-sur-Mer, France), and a hydroxypropyl methylcellulose capsule (HPMC, LGA, La Seyne-sur-Mer, France) were dissolved in lysis buffer (NucliSENS® easyMAG® Lysis Buffer, bioMérieux) at room temperature (RT) in the presence of extraction particles. The extraction particles were then washed, and the captured nucleic acids were eluted. Finally, PCR amplification of the nucleic acids was performed, and detection of the amplified nucleic acids was carried out. The same samples as in Example 4 were tested with the same quantities of NucliSENS® easyMAG® MagSIL extraction particles and the same extraction, amplification, and detection conditions. The PCR results are given in Table 4 in which the REF control corresponds to the Ct obtained with the same protocol implemented without the envelope components.Table 5 shows the difference in Ct between the test performed with a capsule component (data in the first column of Table 4: PVA, Gelatin, or HPMC) and the control or reference (noted Ref LIQUID). This comparison of the amplification results allows us to determine the impact of the capsule component on the performance of the extraction particles. Tables 4 and 5 below show that these capsule shell materials do not affect the performance of the nucleic acid extraction particles, since the differences in Ct (Ct of the tested condition minus the Ct of the reference control) are all within the range of + / - 1 for all capsule components and for all targets (Salmonella, Yersinia, Adenovirus, and Rotavirus) (Table 5).

[0231] Table 4] - Extraction and amplification performance with different envelopes.

[0232] [Table 5] - Difference between the Ct of the liquid reference and the Ct obtained with

[0233] 'envelope. Thus, we can conclude that the nature of these constituents chosen for the envelope of the capsule containing the suspension of magnetic particles does not impact the extraction of nucleic acids and is therefore compatible with the performance of the extraction particles.

[0234] Example 6 - Capsule Stability and Extraction and Amplification Performance (PVA Pouch Containing a Suspension of Magnetic Particles in Glycerol) A 20 mg / L suspension of magnetic particles (NucliSENS® easyMAG® MagSIL, bioMérieux) in 90% w / w glycerol in MES pH 5.5 was packaged. Each sachet of extraction particles suspended in the excipient (glycerol) contains 50 µL, which corresponds to 1 mg of magnetic extraction particles. This water-soluble sachet remained intact for over 3 months, and no leakage of the suspension was observed. This proves that this capsule according to the invention is stable over time and that its contents do not affect the stability and integrity of the capsule shell.

[0235] The sachet was added to 2 ml of a lysis buffer (NucliSENS® easyMAG® Lysis Buffer, bioMérieux) at room temperature, and nucleic acid extraction from various microorganisms in a sample, as described in Example 1, was performed using the eMAG® instrument (BioMérieux, Marcy l'Etoile, France). PCR was then performed as described in Examples 1, 4, and 5 above. The results obtained were as expected and showed no alteration in the performance of the extraction or PCR. It is thus demonstrated that the extraction particles in this capsule according to the invention do not dry out and retain all their nucleic acid-capturing properties, and also that all the components of the capsule, once dissolved and solubilized, do not affect either the extraction or the amplification of nucleic acids by the extraction particles.

[0236] Thus, it will be clear from the above and the examples given that when the capsule comes into contact with water, an aqueous buffer, a lysis buffer, and / or the sample, the capsule's outer shell will dissolve or solubilize, releasing the capsule's contents, namely at least the excipient and the extraction particles. These particles can then come into contact with and capture the biomolecules from the sample being tested. Encapsulation facilitates the handling of the extraction particles. This also allows for precise dosing of the magnetic particles in contact with the sample and optimizes the capture yield. Furthermore, the fact that the extraction particles are contained within a closed internal volume, as defined by the shell, allows for cleaner and more reproducible dispensing of these particles in an automated system.This capsule solution also prevents the need to dilute the particles excessively in a large volume, thus avoiding a loss of stability and therefore capture capacity. It also prevents them from dispersing onto the walls or the sealing film / lid of the extraction device during transport. Using this capsule therefore limits the risk of losing extraction particles or having an extraction cartridge with some particles that are ineffective because they are dry and useless because they are outside the liquid containing the biomolecules to be captured. The particles must always be in contact with a liquid to prevent degradation and maintain their capture performance.

[0237] Furthermore, the fact that the particles are contained within a sealed envelope that also holds a liquid excipient prevents the liquid from evaporating and the extraction particles from drying out during storage or transport. This limits the risk of degradation and therefore the performance loss of particles that are highly susceptible to drying. Indeed, it is essential that the extraction particles not dry out in the container that holds them and then the sample to be tested, or that they not dry out during their manufacturing process, during the manufacturing process of the container in which they will be stored, or during their use in said container that also holds the sample to be tested.

[0238] The capsule according to the invention is easily handled, although it can be flexible, which simplifies the preparation of the container where the test will be performed, the container intended to hold the sample to be tested. Once filled, the capsule according to the invention can thus be easily dispensed into a well of the test container. It therefore provides a solution to the problem of distributing the extraction particles within the container, a distribution that can easily be automated. Furthermore, by its property of confining or compartmentalizing the extraction particles, it also provides a solution to the problem of their dissemination on the walls and / or lid of the container during transport or handling, thereby preventing a reduction in their capture properties (performance) as well as a reduction in the number of particles available to capture biomolecules.It also allows the extraction particles to be contained in a liquid, which maintains their stability and prevents them from drying out, thus degrading their performance. Ultimately, the capsule according to the invention contributes to achieving clean, efficient, optimal, reliable, rapid, and reproducible biomolecule extraction.

[0239] Thus, all the technical problems encountered and mentioned above are solved by the capsule according to the present invention and the processes implementing it, thanks to a selection of the constituents of the envelope, of the excipient allowing the extraction particles to retain all their properties during storage but also during a process of extraction or detection of biomolecules from a sample to be tested.

[0240] Of course, the invention is not limited to the embodiments described and shown in the accompanying figures. Modifications remain possible, particularly with regard to the composition of the various elements or by substitution of technical equivalents, without departing from the scope of protection of the invention.

Claims

DEMANDS 1. Capsule containing particles for extracting biomolecules from a sample to be tested, comprising at least: a closed envelope soluble in an aqueous buffer, a buffer for lysis of cells from the sample to be tested and / or in the liquid sample to be tested, the envelope delimiting an internal volume, particles for extracting biomolecules, preferably silica particles and more preferably magnetic silica particles, an excipient in liquid or viscous form, soluble in an aqueous buffer, a buffer for lysis of cells from the sample to be tested and / or in the liquid sample to be tested, the particles for extracting biomolecules and the excipient being contained in the internal volume delimited by the capsule envelope.

2. Capsule according to claim 1 in which the shell comprises polyvinyl alcohol (PVA); hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose; gelatin, preferably beef, pork or fish gelatin; pullulan; carrageenan or a mixture of at least two of them.

3. Capsule according to any one of the preceding claims wherein the shell comprises at least 80% by weight (w / w) of polyvinyl alcohol (PVA), hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose, gelatin, pullulan, carrageenan or a mixture of at least two of them, preferably at least 90% by weight (w / w) and even more preferably 100% by weight (w / w).

4. Capsule according to any one of the preceding claims, wherein the excipient is selected from DMSO; diethyl formamide; acetonitrile; mono-alcohols, preferably ethanol, butanol, isobutanol, tert-butanol, dimethyl propanol, isopropanol, propanol; di-alcohols, preferably propane-diol, ethylene glycol, triethylene glycol; tri-alcohols, preferably glycerol; vegetable oils, preferably refined almond oil mixed or not with a mono-alcohol, preferably ethanol; a mixture of at least two of these compounds.

5. Capsule according to any one of the preceding claims, wherein the excipient is selected from DMSO, acetonitrile, glycerol and ethanol or a mixture of at least two of them.

6. Capsule according to any one of the preceding claims, wherein the concentration of excipient is at least 40% by weight (w / w).

7. Capsule according to any one of the preceding claims, characterized in that it has an ellipsoid, spherical, cylindrical, cubic, parallelepiped, conical, pyramidal, tetrahedral, octahedral, triangular prism, rectangular or cuboid, pentagonal, hexagonal shape, of a tube, of a capsule, of a bag, of a sachet or of a berlingot.

8. Capsule according to any one of the preceding claims, wherein the shell is made of PVA, gelatin or pullulan and the excipient is glycerol or vegetable oil.

9. Container containing at least one capsule according to one of claims 1 to 8.

10. A method for manufacturing a capsule according to any one of claims 1 to 8 comprising the following steps: Prepare a suspension of biomolecule extraction particles in an excipient or mixture of excipients, Prepare a capsule envelope or use a pre-formed capsule envelope, Fill the internal volume defined by the envelope of the biomolecule extraction particle suspension, Seal the envelope so that it is airtight.

11. A method for extracting biomolecules contained in the sample to be tested comprising: a) A step of contacting the lysed or unlysed sample with the capsule according to one of claims 1 to 8, the solubilized contents of the capsule according to one of claims 1 to 8, or only the extraction particles previously contained in the capsule according to one of claims 1 to 8, b) A lysis step of the sample before and / or after contacting the unlysed sample and the capsule according to one of claims 1 to 8, the contents of the capsule according to one of claims 1 to 8 solubilized or the extraction particles previously contained in the capsule according to one of claims 1 to 8, c) A capture step of the biomolecules of the sample to be tested by the extraction particles, d) Optionally, a washing step of the biomolecules captured by the extraction particles, e) Optionally, an elution step of the biomolecules captured by the extraction particles.

12. Extraction method according to the preceding claim, wherein the solubilization of the capsules is carried out before step a), in step a) or in step b) by the addition of water, an aqueous buffer, a sample lysis buffer, by the sample, by shearing, by agitation, by abrasion, by heating, by ceramic beads or an element configured to at least weaken, crack or break the capsule shell, or by a combination of at least two of them.

13. Extraction process according to any one of claims 11 or 12, wherein the extracted biomolecules are nucleic acids.

14. A method according to any one of claims 11 to 13, characterized in that it further comprises: Optionally, a nucleic acid amplification step when the biomolecules extracted at the end of the process according to any one of claims 11 to 13 are nucleic acids, preferably the amplification is carried out by PCR, A step in detecting biomolecules.

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