Method for solid-phase extraction using a porous monolith

Abstract

The invention relates to a method for solid-phase extraction and / or separation of one or more compounds of interest from a liquid sample which comprises: • - integrating a self-supporting porous monolith (20) in a fluid duct (30) such that the self-supporting porous monolith (20) is stationary in the fluid duct (30) during said method and forms a filter in the fluid duct; • - passing the sample (300) at least once through the porous monolith in the fluid duct (30) over at least one portion of the porous monolith (20), the self-supporting porous monolith (20) having a largest dimension less than or equal to 3 mm transverse to the fluid duct (20), the fluid duct having one or more open ends prior to integration, the or at least one of the ends remaining open during the step of integrating the porous monolith.

Description

[0001] Description

[0002] Title: Solid phase extraction process using a porous monolith

[0003] The present invention relates to a method for solid phase extraction of one or more compounds of interest using a porous monolith integrated into a fluid conduit. The invention also relates to a method for integrating a porous monolith into a tube made of a heat-shrinkable material.

[0004] Technical field

[0005] The extraction of one or more compounds of interest from a complex mixture is essential for subsequent use, particularly to enable their identification and / or quantification. The quality of the extraction of the compound(s) of interest has a significant impact on the quality of the results obtained subsequently.

[0006] There are currently several methods for extracting compounds of interest.

[0007] A practical and efficient way to perform an extraction is to circulate a solution containing the molecules of interest through a solid phase. For this, it is necessary to have a fluidic circuit incorporating a porous material of a cross-section perfectly adapted to the conduit in which it is integrated and fixed to ensure that the solution circulating in the circuit inevitably passes through the porous material.

[0008] Among the materials that can be used as solid phase for extraction, porous monoliths, especially those with hierarchical porosity (HPM), have advantages such as good permeability, porosity and tunable surface chemistry. The realization of devices for extraction in a miniaturized format is a challenge.

[0009] As described in the articles by Nema et al., Talanta 82 (2010) 488-494 Application of silica-based monolith as solid phase extraction cartridge for extracting polar compounds from urine, Ma, X., Zhao, M., Zhao, F. et al. Application of silica-based monolith as solid-phase extraction sorbent for extracting toxaphene congeners in soil. J Sol-Gel Sci Technol 80, 87-95 (2016) and Jorge C. Masini, et al., Porous monolithic materials for extraction and preconcentration of pollutants from environmental waters, Trends in Environmental Analytical Chemistry, Volume 29, 2021, e00112, ISSN 2214-1588, one method for integrating an HPM into a fluidic circuit is to embed it in the body of a syringe, in particular by force insertion. This is only possible for HPMs with sufficient resistance to manual embedding constraints, particularly those several millimeters wide.As described in application JP2003166983, another method for encapsulating an HPM for SPE or chromatography use is to use a heat-shrinkable tube which, when heated to 100°C for 10 minutes, shrinks onto the HPM to encapsulate it and hold it in position. This method is commonly used to encapsulate porous monoliths several mm wide. However, for porous monoliths of small diameter, in particular with a diameter of less than or equal to 2 mm, it is established, in particular in US patent 7,291,383, that the encapsulation as described in the aforementioned Japanese patent application does not make it possible to maintain both the integrity of the HPM and the tightness of the integration.

[0010] Such extraction devices with large HPM widths require several hundred microliters to be circulated several times over the HPM. They operate with flow rates obtained by creating an air vacuum at the device outlet or by centrifugation. They are therefore not very modular and difficult to control precisely. In addition, they are not always compatible with the constraints of certain fields, particularly so-called -omics fields including proteomics, metabolomics or glycomics, in terms of reduced sample quantity, small volume of samples to be analyzed and required speed.

[0011] A solution proposed to miniaturize such systems, notably by US patent 7,291,383 and the article by Jorge C. Masini, et al., Porous monolithic materials for extraction and preconcentration of pollutants from environmental waters, Trends in Environmental Analytical Chemistry, Volume 29, 2021, e00112, ISSN 2214-1588 is to form the HPM in situ in a capillary with an internal diameter of less than 1 mm. In the capillaries obtained, porosity gradients exist, as shown in particular in the article by Bruns, S., Milliner, T., Kollmann, M., Schachtner, J., Hôltzel, A., & Tallarek, U. (2010). Confocal laser scanning microscopy method for quantitative characterization of silica monolith morphology.Analytical chemistry, 82(15), 6569-6575, which affects reproducibility, and scanning electron microscopy images often show on the one hand that the anchoring of HPMs in the capillaries is incomplete, releasing interstitial spaces that can affect reproducibility or even the quality of extraction by creating preferential paths and on the other hand, that for the same formulation, the structure is not similar when the diameter of the capillary varies, which implies that major modifications are made to the initial compositions but also to the process steps compared to what is done for larger self-supporting monoliths to limit in particular the edge effects at the level of the capillary wall, which makes the preparation of such capillaries long and complex and leads to structural variations.

[0012] International application WO 2005 / 072164 describes a monolith integrated into a polyethylene tube by inserting the monolith into the tube, then the assembly is heated until the tube is in a molten state and encapsulates the monolith, and finally the ends are cut to allow the circulation of fluid in the porous monolith. International application WO 2008 / 112702 describes a system comprising a porous monolith integrated into a support by heating the support to shrink it onto the porous substrate. This document describes more particularly a porous monolith of 1.2 mm in diameter inserted into a borosilicate glass tube having an internal diameter of 1.8 mm and heating the assembly to a temperature of 850°C to melt the glass and shrink it onto the monolith, then cutting the assembly to obtain parts usable in chromatography.Putting the tubes in a molten state causes the molten material to penetrate into the pores of the porous monolith over its entire surface. It is therefore necessary to cut the assembly to allow access to the porous part of the monolith. Such a cut makes the use of the assembly more complex, particularly in terms of connection, and can damage the porous monolith, especially in the case of a small diameter monolith. In addition, the penetration of the tube material into the pores of the monolith makes the use of small diameter monoliths complicated, as there is a risk of filling all the pores of the monolith when the tube material melts, thus blocking the porous monolith.

[0013] There is therefore a need for a method for extracting compounds of interest from small volume samples that is simple, rapid, efficient and reproducible.

[0014] Statement of the invention

[0015] The invention meets this need using a method for solid phase extraction of one or more compounds of interest from a liquid sample comprising: the integration of a self-supporting porous monolith in a fluid conduit so that the self-supporting porous monolith is fixed in the fluid system during said method and forms a filter in the fluid system, at least one passage of the sample through the porous monolith in the fluid conduit over at least a portion of the porous monolith, the self-supporting porous monolith having a largest transverse dimension to the conduit less than or equal to 3 mm.

[0016] By "forms a filter", we mean that any liquid passing through the fluid conduit passes through the porous monolith over at least part of its length, in particular over its entire length, the length being defined as the largest dimension along the axis of the fluid conduit. The fact that the porous monolith is of a dimension less than or equal to 3 mm makes it possible to process samples of very small volumes, which is particularly interesting in certain fields but also allows for rapid and robust processing. This also makes it possible to optimize the subsequent analysis of the extracted compounds of interest by reducing the need for preconcentration of the latter before their analysis.

[0017] The use of a porous monolith allows for a device with very good extraction efficiency and which is versatile. It is in fact easy to adapt the porosity characteristics and the surface chemistry of the porous monolith to the type of extraction required.

[0018] Monolith formation process

[0019] Preferably, the porous monolith is formed by a sol-gel process. By "sol-gel process" is meant a process implemented using as precursors alkoxys of formula M(OR)n, R'-M(OR)nl or sodium silicates or titanium colloids, M being a metal, a transition metal or a metalloid, in particular silicon, and R or R' alkyl groups, n being the oxidation state of the metal. In the presence of water, hydrolysis of the alkoxy groups (OR) occurs, forming small particles generally less than 1 nanometer in size. These particles aggregate and form clusters that remain in suspension without precipitating, and form the sol. The increase in clusters and their condensation increases the viscosity of the medium and forms what is called the gel. The gel can then continue to evolve during an aging phase during which the polymer network present within the gel becomes denser.The gel then shrinks, evacuating the solvent outside the formed polymer network, during a step called syneresis. Then the solvent evaporates, during a step called drying, which leads to a solid material of the porous glass type giving a porous monolith. The syneresis and drying steps can be concomitant.Preferably, the self-supporting porous monolith is formed by a manufacturing method comprising: forming a sol comprising a sol-gel precursor in aqueous solution and, preferably, a pore-forming agent, at least partially filling an enclosure and at least one mold contained in the enclosure with previously formed sol, the mold comprising at least one opening opening into the sol after filling with sol, forming a sol-gel matrix in the enclosure from the sol, extracting the mold with the sol-gel matrix contained in the mold from the enclosure, and extracting the sol-gel matrix from the mold, forming a porous monolith from the sol-gel matrix extracted from the mold, the formation of the sol, the sol-gel matrix and the porous monolith being carried out by a sol-gel process.

[0020] The presence of at least one opening in the mold below the soil level after filling allows the mold to be filled with soil during the filling step and the fluid circulation of the soil between the soil contained in the mold and the soil contained in the enclosure during the rest of the process. The fact of producing a large sol-gel matrix in the enclosure and extracting a part of it included in a mold during the formation of the matrix makes it possible to overcome the edge effects that appear in the processes described previously by producing the sol-gel matrix in a container that is always the same size. Such a process allows the manufacture of self-supporting porous monoliths with similar textural properties over a wide range of diameters without having to reoptimize, or even modify, the formulation of the initial mixture.It also allows access to a wide variety of shapes and aspect ratios of the monoliths, but also to varied and reproducible controlled internal structures. The structures obtained are particularly uniform and therefore guarantee homogeneity of resistance to mechanical stresses. This can be useful, for example, to avoid breakages when pressure is exerted on the monolith during its integration into the fluidic conduit, in particular during its encapsulation in a heat-shrinkable fluidic conduit. The sol can be formed by stirring a solution comprising the sol-gel precursor, preferably the sol-gel precursor and the pore-forming agent, in particular for a duration greater than or equal to 5 min, better still greater than or equal to 10 min, even better greater than or equal to 15 min. The duration of stirring can be less than or equal to 3 h, better still less than or equal to 2 h.During stirring, the temperature can be controlled at a substantially constant predetermined value, in particular between 0°C and 90°C, better between 0°C and 50°C.

[0021] Filling can be done without the presence of air bubbles and / or gradients in chemical composition and / or temperature of the soil in the enclosure and the mold(s).

[0022] The formation of the sol-gel matrix may involve condensation to form a gel and optionally at least partial aging to densify the gel.

[0023] Preferably, the formation of the sol-gel matrix in the enclosure is without drying of the sol-gel matrix.

[0024] The formation of the sol-gel matrix can be done in the same way in the enclosure and the mold. The total porosity and the pore size are preferably substantially homogeneous in the enclosure and the mold(s).

[0025] The extraction of the mold with the matrix it contains from the enclosure may comprise an extraction of a block of the sol-gel matrix containing the mold from the enclosure and the extraction of the mold and the sol-gel matrix it contains from the previously extracted block. The extraction of the or each mold from the block may be done by cutting the sol-gel matrix flush with the corresponding mold or breaking the sol-gel matrix surrounding the mold(s). Alternatively, the extraction of the or each mold with the matrix it contains may be done by removing the corresponding mold from the sol-gel matrix surrounding it after extraction of the block as described above or directly in the enclosure without prior extraction of the block, in particular when the corresponding mold is only partially immersed in the sol-gel matrix.

[0026] The extraction of the sol-gel matrix contained in the or each mold can be done by means of controlled pressure on said sol-gel matrix, for example by direct pressure with a solid of smaller size than the mold or by pressure of a gas at controlled flow rate or by opening the or each mold, in particular by cutting the or each mold or separating two parts of the or each mold from each other. The mold or molds can be in the form of two parts that are movable between them, in particular separable or movable relative to each other by a hinge. The mold containing the sol-gel matrix can be immersed in a liquid during the step of extracting the sol-gel matrix contained in the mold. This facilitates the extraction of the sol-gel matrix.

[0027] The method for manufacturing the porous monolith may comprise a controlled generation of mesoporosity in the sol-gel matrix to form a sol-gel matrix with hierarchical porosity after the mold is extracted from the enclosure, and before the formation of the porous monolith from the sol-gel matrix of the mold. The controlled generation of mesoporosity may be carried out by immersing the sol-gel matrix, whether or not extracted from the or each mold, in an aqueous mesoporosity-generating solution comprising a sol-gel matrix dissolution agent and / or a sol-gel matrix dissolution agent precursor. The dissolution agent may be ammonium hydroxide, for example at a 1M concentration, sodium hydroxide, or hydrofluoric acid or mixtures thereof. The sol-gel matrix dissolution agent precursor may be urea or compounds bearing amide functions, in particular formamide, acetamide, N-methylformamide (NMF) and mixtures thereof.Preferably, the concentration of dissolving agent and / or dissolving agent precursor is such that it allows the localized dissolution of the sol-gel matrix(es) so as to form mesopores in the latter without dissolving the sol-gel matrix(es) overall.

[0028] The formation of the porous monolith may involve at least partial aging to densify the sol-gel matrix, particularly when aging has not completely occurred before.

[0029] The formation of the porous monolith may involve drying the sol-gel matrix, whether or not extracted from the mold, to form a dried sol-gel matrix, after the generation of mesopores if necessary. The drying step may be carried out under a flow of air or inert gas, in particular nitrogen, argon or carbon dioxide, helium, or even oxygen or hydrogen.

[0030] The formation of the porous monolith may include a heat treatment of the sol-gel matrix(es) extracted or not from the or each mold, in particular after drying. The heat treatment may be carried out in a closed container under a flow of air or inert gas, in particular nitrogen, argon or carbon dioxide, helium, or even oxygen or hydrogen, and by gradual heating followed by maintaining the final temperature for a predetermined time. The gradual heating may be an increase of 0.5°C / min until reaching a temperature greater than or equal to 300°C, better still greater than or equal to 340°C, for example substantially equal to 350°C, to obtain a porous monolith. The final temperature may be maintained for more than 1 hour. This makes it possible to stabilize the structure of the monolith and to eliminate the organic residues resulting from the synthesis.

[0031] Alternatively, the porous monolith is formed directly in a mold into which the soil is inserted and extracted from the latter, in particular by the method mentioned above.

[0032] Porous monolith

[0033] The porous monolith may comprise macropores, in particular macropores with a dimension greater than or equal to 50 nm. The macropores may have a dimension less than or equal to 30 μm. The porous monolith may comprise mesopores, in particular with a dimension less than or equal to 50 nm, better between 2 and 50 nm. Preferably, the pores are connected to each other in the porous monolith. This makes it possible to obtain porous monoliths with hierarchical porosity, that is to say having at least two orders of magnitude of pore sizes, preferably macropores formed during the formation of the sol-gel matrix and mesopores formed during the controlled generation of mesopores. Such porous monoliths have a good exchange surface between the solution passing through it and its material and minimizes the distances to be covered by diffusion. In addition, this makes it possible to provide flexibility to the sol-gel matrix while reducing the risks of breakage.In addition, this helps to reduce the drying time of the sol-gel matrix.

[0034] The porous monolith may have a larger transverse dimension to the conduit less than or equal to 2 mm, better still less than or equal to 1.5 mm, even better still less than or equal to 1 mm.

[0035] The self-supporting porous monolith may have a larger transverse dimension to the conduit greater than or equal to 20 pm.

[0036] Preferably, the porous monolith may have a length greater than or equal to 0.5 mm, better still greater than or equal to 1 mm, even better still greater than or equal to 2 mm.

[0037] Preferably, the porous monolith may have a length, taken along the longitudinal axis of the conduit, greater than its largest transverse dimension.

[0038] Preferably, the porous monolith is integrated into the conduit and configured such that any liquid passing through the fluid conduit passes through the porous monolith for a distance of at least 10% of its length, more preferably at least 50%, and most preferably the entire length.

[0039] Preferably, the porous monolith is cylindrical with a diameter less than or equal to 1.5 mm, better still less than or equal to 1 mm, and a length greater than or equal to 0.5 mm, better still greater than or equal to 1 mm, even better still greater than or equal to 2 mm.

[0040] The porous monolith can be of substantially homogeneous structure throughout its volume.

[0041] The porous monolith may have an aspect ratio, defined as the ratio of its height to its largest transverse dimension, greater than or equal to 0.2, better still greater than or equal to 0.4, better still greater than or equal to 1 and / or less than or equal to 1000, better still less than or equal to 500, even better still less than or equal to 100, even better still less than or equal to 50, even better still less than or equal to 20.

[0042] Preferably, the porous monolith is cylindrical with a polygonal, oval or circular base, in particular cylindrical of revolution.

[0043] The process may include post-manufacturing modifications of the porous monolith, including functionalization of the surface of the porous monolith, especially before or after its integration into the conduit. The surface of the porous monolith may be coated with molecules such as hydrophobic hydrocarbon ligands (e.g., octadecyl ligands) or hydrophilic ligands such as 2,3-dihydroxypropyl derivatives. The ligands of such modified columns may be further modified using known procedures. Porous catalysts or enzyme supports may be prepared by adding enzymes, e.g., glucose isomerase, peptide-N-glycosidase F (PNGase F), or catalytic metal elements, e.g., platinum and palladium.

[0044] Fluid conduit

[0045] Preferably, the fluid conduit has at least one open end, better still two open ends.

[0046] Preferably, the fluid conduit is through. The fluid conduit is preferably open on either side of the porous monolith.

[0047] Alternatively, the fluid conduit is closed at its end downstream of the porous monolith so as to form a well. In this case, the porous monolith may be at the closed end of the conduit or the fluid conduit may have a space between its closed end and the porous monolith. The sample may then pass through the porous monolith from one end to the other during its passage through the porous monolith, in particular when a space is present downstream of the latter, or pass through it over part of its height in a round trip, in particular in the case where the porous monolith is at the closed end of the conduit.

[0048] Preferably, the fluid conduit is single-layer.

[0049] The fluid conduit may be made of a heat-shrinkable polymer, in particular polyolefin, such as polyethylene (PE) or polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP), polyetheretherketone (PEEK), or polyvinylidene fluoride (PVDF) or a mixture thereof.

[0050] The fluid conduit may have a wall of constant thickness greater than or equal to 0.004 mm, better still greater than or equal to 0.15 mm, or even better still 0.2 mm. Preferably, the thickness of the wall of the conduit is less than or equal to 1 mm, better still less than or equal to 0.9 mm, even better still less than or equal to 0.65 mm.

[0051] The fluid conduit can be rigid, flexible, or preferably semi-rigid.

[0052] The fluid conduit can have an elastic wall.

[0053] The fluid conduit can be transparent. This allows direct visualization of the passage of the solution, especially when the latter is colored, which allows monitoring of the process.

[0054] Alternatively, the conduit may be completely or at least partially opaque. The conduit may have a transparent viewing window at the porous monolith.

[0055] Preferably, the fluid conduit is configured to have, after integration of the porous monolith, a minimum diameter less than or equal to, better strictly less than, the largest transverse dimension of the porous monolith, preferably the smallest transverse dimension of the porous monolith, in particular its diameter, the transverse dimension being understood along a transverse plane of the conduit after integration of the monolith in the conduit. The fluid conduit may have a minimum diameter strictly less than 100%, better less than or equal to 98%, even better less than or equal to 95%, of the smallest transverse dimension of the porous monolith, in particular its diameter. The minimum diameter of the conduit may be greater than or equal to 50%, better 60%, better 70%, better 80%, of the smallest transverse dimension of the porous monolith.The fluid conduit may have a shrinkage coefficient, defined as the ratio between the initial diameter and the minimum diameter achievable after shrinkage, greater than or equal to 1.5:1, better still greater than or equal to 2:1. Preferably, the shrinkage coefficient is less than or equal to 6:1.

[0056] The fluid conduit may be configured to shrink at a temperature greater than or equal to 70°C, better still greater than or equal to 100°C.

[0057] Alternatively, the fluid conduit may be a pipette tip or a solid phase extraction cartridge. In this case, the porous monolith may be force-fitted into the fluid conduit. The fluid conduit may have an inner wall having a sealing layer that comes into contact with the outer wall of the porous monolith. Such a layer may be an adhesive layer or a layer of an elastic material. The fluid conduit may have an inner diameter less than or equal to that of the porous monolith. In this case, the fluid conduit and the porous monolith may be of complementary conical shape. Preferably, at least a portion of the conduit of a length greater than or equal to the length of the porous monolith, better still the entire conduit, is in the form of a cylindrical tube, in particular with a circular base.Alternatively, at least a portion of the conduit of length greater than or equal to the length of the porous monolith, better still the entire conduit, is of truncated conical shape. By "length of the porous monolith" is meant its largest dimension along the longitudinal axis of the conduit after integration. Preferably, the porous monolith has an external surface of the same shape as said portion of the conduit, within the dimensions where appropriate.

[0058] Preferably, the fluidic conduit has, after integration of the porous monolith, a fluid circulation length strictly greater than the length of the porous monolith. Preferably, the fluidic conduit has at its or one of its open ends, after integration of the porous monolith, a non-zero length of conduit devoid of porous monolith. This makes it easier to introduce the sample into the tube upstream of the porous monolith by facilitating the integration of the assembly into the extraction device by the ease of fluidic connection of the extended end. In addition, the fluidic circulation length can be adapted according to the specific fluidic protocol of a particular use case.

[0059] Preferably, the porous monolith is integrated into the fluid conduit without protruding from the fluid conduit. This facilitates the integration of the fluid conduit into a fluid device and facilitates the passage of the liquid sample into the fluid conduit. This also makes it possible to have a reserve of liquid in the fluid conduit, in particular upstream of the porous monolith, which can make it possible to prepare in advance a quantity of a solution or a sequence of several solutions in predetermined quantities in the fluid conduit upstream of the porous monolith to be passed through the porous monolith.

[0060] Integration stage

[0061] Preferably, the fluid conduit has one or more open ends before integration of the porous monolith and the or at least one of the open ends of the fluid conduit before integration remains open during integration of the porous monolith. This allows passage of the sample through the porous monolith without it being necessary to open one end of the conduit after integration of the porous monolith.

[0062] Preferably, the porous monolith retains its integrity during the integration step in the fluidic conduit. In particular, the integration of the porous monolith in the fluidic conduit is done so that it is not necessary to cut a part of the porous monolith, in particular one of its ends to allow the passage of the sample therein. This makes it possible to limit the risk of degradation of the porous monolith by limiting the actions on the latter and facilitates the integration of the entire fluidic conduit integrating the porous monolith in a sample circulation device. In particular, during the integration step, the fluidic conduit is not melted to encapsulate the porous monolith.Such integration would generate a local penetration of the material of the fluidic conduit on the surface of the porous monolith, which would require, to allow the passage of the sample, to cut, at least at one end of the porous monolith, the porous monolith over the penetration thickness, which could degrade the porous monolith, in particular when it is of small diameter.

[0063] Preferably, the integration of the porous monolith is done without it being necessary to amputate the fluid conduit of a part of its length to allow the passage of the sample through the porous monolith. The resulting device makes it possible to limit the steps necessary before the passage of one or more solutions, in particular the sample, into the fluid conduit.

[0064] Preferably, the fluid conduit is made of a heat-shrinkable polymer and integrating the porous monolith into the fluid conduit may comprise inserting the porous monolith into the fluid conduit and heating the fluid conduit to shrink the conduit so as to encapsulate the porous monolith within said conduit.

[0065] Preferably, the heating of the conduit is carried out at a temperature greater than or equal to the minimum shrinkage temperature of the conduit, in particular at a temperature greater than or equal to 70°C, better still greater than or equal to 100°C, even better still greater than or equal to 300°C. Preferably, the heating of the conduit is carried out at a temperature less than or equal to the melting or degradation temperature of the fluid conduit, in particular less than or equal to 800°C, preferably less than or equal to 700°C, better still less than or equal to 500°C.

[0066] Preferably, the shrinkage of the conduit is such that, after shrinkage, the inner wall of the conduit extends along the envelope surface of the porous monolith without merging into the surface pores of the porous monolith.

[0067] Preferably, the retraction of the conduit is such that the porous monolith remains fixed in the conduit when a solution is introduced into the tube at a pressure of between 0.05 and 7 bars, preferably between 0.1 and 5 bars, even better between 0.15 and 4 bars.

[0068] Preferably, the heating of the conduit is carried out by heating the conduit for a duration and at a temperature chosen so that the fluid conduit shrinks until it comes into contact with the external wall of the porous monolith over at least part of the length of the porous monolith, better still over the entire length of the porous monolith, in particular until it matches the shape of the envelope surface of the porous monolith. Preferably, after heating, the portions of the conduit directly upstream and downstream of the porous monolith have respective diameters strictly less than 100%, better still less than or equal to 95%, better still 90% of the smallest transverse dimension of the end section of the porous monolith directly adjacent.

[0069] The heating of the duct can be carried out using a heating gun moved manually along the duct. Preferably, the heating gun is moved along the duct in a substantially regular manner so as not to heat a portion of the duct for more than 10 minutes, better still more than 2 minutes, even better than 1 minute. The heating gun can be set to emit an output temperature of more than 450°C during the heating of the duct and be positioned less than 15 cm, better still less than 4 cm, even better still 2 cm from the duct during heating. Alternatively, the heating is carried out in an oven. In this case, the oven temperature is at least 70°C, better still greater than or equal to 150°C, even better still greater than or equal to 300°C, preferably less than or equal to 800°C, better still less than or equal to 700°C, even better still less than or equal to 500°C.Preferably, it is chosen between the minimum shrinkage temperature and the melting or degradation temperature of the fluid conduit. The heating can be maintained for a temperature-dependent duration so that the heat-shrinkable polymer of the fluid conduit does not degrade, in particular less than or equal to 1 h for a temperature between 300 and 450°C and less than or equal to 30 min, better still 15 min for a temperature between 450°C and 650°C.

[0070] Preferably, the entire conduit and porous monolith after integration is characterized in that it is devoid of a continuous fluid path extending between the internal wall of the conduit and the porous monolith connecting portions of conduit over lengths at least 20 times greater than the average size of the macropores, better still over lengths at least 10 times greater than the average size of the macropores.

[0071] Alternatively, the heating step may be carried out by a predetermined variable temperature profile. The predetermined temperature profile may include temperature steps and / or a gradual temperature increase ramp.

[0072] The method may include integrating a plurality of porous monoliths into a plurality of disconnected conduits and passing a plurality of samples, each through a porous monolith in one of the conduits.

[0073] The porous monolith may be integrated such that, during passage of the sample through the fluid conduit, any fraction of the sample flows through the porous monolith over at least a portion of the length of the latter.

[0074] After integration of the porous monolith into the fluidic conduit, the fluidic conduit can be integrated into a fluidic device configured to introduce a solution, in particular the sample, into the conduit and circulate it.

[0075] The fluidic device may comprise a solution supply member configured to be connected to one end of the fluidic conduit. The liquid supply member may be configured to be connected at its other end to an interchangeable liquid reservoir depending on the solution to be introduced into the conduit, in particular to a reservoir containing the sample. The fluidic device may comprise a pressure controller or a syringe pump connected to the fluidic conduit upstream or downstream of the porous monolith, in particular via the liquid supply member of the conduit, configured to control the circulation of the fluid in the conduit.

[0076] Alternatively, the device may comprise a centrifugation device for passing a solution, in particular the sample, through the porous monolith by centrifugal force, in particular when the conduit has a closed end.

[0077] The conduit may be open at both ends and the fluidic device may comprise a liquid distribution member at the outlet of the fluidic conduit. The liquid distribution member may be a needle. Such a liquid distribution member may facilitate the accuracy of liquid recovery at the outlet of the conduit and the accuracy of distribution onto or into one or more sampling members. Alternatively, the solution at the outlet of the conduit is directly recovered without passing through an additional distribution member at the outlet of the conduit.

[0078] Sample

[0079] Preferably, the sample passing through the porous monolith is less than 500pL, better less than 250pL, even better less than 100pL.

[0080] The sample may be formed from a blood sample, including plasma or serum, cerebrospinal fluid, urine or milk, including human. Preferably, such a sample is modified before passing through the porous monolith.

[0081] Solid phase extraction

[0082] The method may be a solid phase extraction method of compounds of interest by adsorption of the latter on the surfaces of porous monoliths to separate them from the rest of the sample, then recovery by elution of the compounds of interest adsorbed on the surfaces of the porous monolith. The method preferably comprises, before the sample is passed, the conditioning of the porous monolith by passing one or more successive conditioning solutions in the fluidic conduit through the porous monolith. Such conditioning makes it possible to impregnate the porous monolith with solvent in order to improve the adsorption of the compounds of interest. The conditioning of the porous monolith in the conduit may comprise the passage of a polar solvent, in particular comprising water and / or acetonitrile. Preferably, the polar conditioning solvent is identical to the solvent of the sample.The conditioning may include the passage of a first water solution, in particular pure water, into the conduit through the porous monolith and the passage of an aqueous acetonitrile solution, in particular at 80% by volume.

[0083] The method may include, after the sample has passed through the porous monolith, washing the porous monolith by passing one or more washing solutions in the fluid conduit through the porous monolith. The washing solution may be of lower polarity than the sample solvent. The washing solution may be an aqueous solution of acetonitrile at more than 50% by volume, better still 70%. Such washing makes it possible to remove impurities from the porous monolith without eluting the compounds of interest in order to improve the purity of the analytes.

[0084] The method may comprise, after the sample has been passed and, where appropriate, after washing, the elution of the compounds of interest by passing an eluent solution or several fractions of eluent solution through the porous monolith in the fluid conduit and the recovery of the eluate(s) in particular for the purpose of analyzing them to determine their composition in compounds of interest. The elution may be carried out by passing several successive fractions, disjointed or not, of eluent solution through the porous monolith and by recovering each eluate after its passage through the porous monolith in dissociated recovery systems. The or each fraction of eluent solution may have a volume less than or equal to 10pL, better still less than or equal to 5pL, even better still less than or equal to 1pL.The recovery of eluates can be done on different areas of a plate, in particular on a mass spectrometer plate using a matrix-assisted laser desorption / ionization (MALDI) source coupled or not to a time-of-flight analyzer (MALDI-TOF).

[0085] The method may include analysis of the eluate(s). The eluent solution may be of higher polarity than the sample solvent, such as pure water.

[0086] The analysis can be carried out by mass spectrometry, in particular a mass spectrometer coupling a matrix-assisted laser desorption / ionization (MALDI) source and a time-of-flight (TOF) analyzer, by liquid chromatography coupled with fluorescence detection (LC-Fluo) or by capillary electrophoresis coupled with fluorescence (CE-LIF). The analysis is preferably carried out after recovery of the eluent solution, possibly after addition of a matrix substance adapted to the analysis method, in particular MALDI-TOF, in particular a solution comprising 2,5-dihydroxybenzoic acid in 50% by volume methanol, without a prior step of drying the eluent solution.

[0087] Preferably, the extraction process is carried out in the absence of acid, in particular formic or trifluoroacetic acid. Alternatively, an acid, in particular a proportion greater than or equal to 0.1% by volume of an acid, in particular formic or trifluoroacetic acid, is added to the conditioning solution, the washing solution and / or the eluent solution.

[0088] A base can be added to the conditioning solution, the washing solution and / or the eluent solution.

[0089] Preferably, the extraction method comprises a preliminary step of preparation in the fluid conduit upstream of the porous monolith of a sequence of the different successive solutions which must pass through the porous monolith to allow extraction, in particular of the conditioning solution, the sample, the washing solution and the eluent solution in the form of one or more successive fractions. This allows precise control of the extraction by simply controlling the pressure in the fluid conduit. This also allows precise control of the volumes of the different solutions and the times between the different steps.

[0090] The process may involve a single passage of the sample and / or the different solution(s) through the porous monolith. In this case, the sample and / or the solution(s) preferably pass through the porous monolith along its entire length. The conduit is then preferably open on either side of the porous monolith or has a free space downstream of the porous monolith.

[0091] Alternatively, the method may include passing the sample and / or the different solution(s) through the porous monolith more than once along part or all of its length. The sample and / or the different solution(s) may thus make one or more round trips through a portion or all of the porous monolith. This is particularly the case when the porous monolith is at the bottom of a closed conduit.

[0092] Preferably, the passage of the sample and / or the different solution(s) through the porous monolith is forced in particular by controlling the pressure in the fluid conduit or by any other means, in particular by applying a centrifugal force. Alternatively, the passage of the sample and / or the different solution(s) through the porous monolith may be done by gravity.

[0093] In particular in the case of a porous monolith integrated into the bottom of a fluid conduit closed at one of its ends, the method may include stirring the fluid conduit to allow the circulation of the solution and / or the sample present in the porous monolith at each of the steps of passage of a solution through the porous monolith mentioned above.

[0094] The method may be a method of extracting free N-glycans or oligosaccharides from a biological sample.

[0095] Preferably, the sample passing through the porous monolith comprises free N-glycans or oligosaccharides and the porous monolith is configured to adsorb these species contained in the sample as the latter passes through the porous monolith.

[0096] The method may include sample preparation prior to extraction, in particular to release N-glycans or oligosaccharides into the sample. In the case of N-glycan extraction, the sample may be prepared prior to extraction according to the method described in the article by Sophie Cholet, et al. N-glycomics and N-glycoproteomics of human cerebrospinal fluid. Current Proteomic Approaches Applied to Brain Function, 127, Humana Press, 360 p., 2017, Neuromethods, 978-1-4939-7119-0 978-1-4939-7118-3.The sample preparation may include the following steps, in particular as described in the aforementioned article: the release of N-glycans by denaturation of the glycoproteins followed by enzymatic deglycosylation of the proteins and the conversion into glycans of the residual glycosylamines in the solution obtained after deglycosylation of the proteins, derivatization by permethylation as described in the article by Sophie Cholet et al mentioned above or better by ethyl esterification of the sialic acids of the N-glycans of the solution obtained after deglycosylation, as described in F article Reiding et al, Anal. Chem. 86, 5784-5793 (2014) High-Throughput Profiling of Protein N Glycosylation by MALDI-TOF-MS Employing Linkage-Specific Sialic Acid Esterification .

[0097] The process may involve adding a polar solvent to the sample, such as an 80% aqueous polar solvent of acetonitrile, prior to passing the sample through the porous monolith. Recyclability

[0098] The method may include recycling the porous material by heat treatment. The heat treatment may be carried out by retaining the porous monolith in the fluid conduit. Alternatively, the recycling heat treatment is carried out after the porous monolith has been extracted from the fluid conduit. The heat treatment may be carried out by heating the porous monolith to a temperature greater than or equal to 100°C, better still greater than or equal to 150°C, even better still greater than or equal to 200°C for a duration greater than or equal to 0.5 h, even better still greater than or equal to 1 h.

[0099] The process may include reuse after recycling of the porous monolith.

[0100] Encapsulation process

[0101] The invention also relates, according to another aspect, to a method for integrating a self-supporting porous monolith into a heat-shrinkable conduit, in particular made of polymer, comprising:

[0102] The integration of the porous monolith into the conduit,

[0103] Shrinking the conduit by heating the latter according to a predetermined temperature profile over time, the temperature profile over time being configured so that the porous monolith is held fixedly in the conduit and the force applied to the self-supporting porous monolith is less than the limit resistance force that a test porous monolith manufactured by the same process as said self-supporting porous monolith, of the same length as said self-supporting porous monolith and of greatest transverse dimension less than or equal to 3 mm would have.

[0104] By "limit resistance force that a porous monolith would have" we mean the force that must not be exceeded in order not to damage the porous monolith.

[0105] Such an encapsulation process allows all sizes of porous monolith to be encapsulated without damaging it, including porous monoliths less than 3 mm in diameter.

[0106] Preferably, the porous monolith has a largest transverse dimension to the conduit less than or equal to 3 mm, better than 2 mm, even better than 1.5 mm.

[0107] The characteristics of the porous monolith, the fluidic conduit and the method for integrating the porous monolith described above apply independently of the extraction method. The invention also relates to a method for solid-phase extraction of one or more compounds of interest from a liquid sample comprising: the integration of a self-supporting porous monolith in a fluidic conduit such as according to the previous aspect of the invention, at least one passage of the sample through the porous monolith in the fluidic conduit.

[0108] The characteristics described above in relation to the preceding extraction method apply independently of said extraction method.

[0109] Gly comic analysis method

[0110] The invention also relates to a method for glycomic analysis of a sample containing N-glycans comprising:

[0111] At least one passage of the sample through a preconditioned porous monolith to adsorb the N-glycans of the sample, the porous monolith being integrated into a conduit of a fluidic device,

[0112] Washing the porous monolith by passing a washing solution through the porous monolith,

[0113] Elution of N-glycans by passing an eluent solution through the porous monolith,

[0114] Sampling of the eluent solution at the outlet of the porous monolith, Glycomic analysis of the eluent solution sampled.

[0115] The characteristics described above in relation to the preceding extraction method apply independently of said extraction method when they are compatible.

[0116] Brief description of the drawings

[0117] [Fig 1] schematically represents an example of an extraction device making it possible to implement the extraction method according to the invention,

[0118] [Fig 2] is a section along II-II of the entire conduit and the porous monolith integrated into the conduit of figure 1,

[0119] [Fig 3] is a view of a detail according to III of figure 2,

[0120] [Fig 4] represents a method for manufacturing a porous monolith allowing the implementation of the method according to the invention, [Fig 5] represents different stages of a solid phase extraction method according to the invention,

[0121] [Fig 6] represents the step of recovering the eluent solution at the outlet of the conduit after the implementation of the extraction process,

[0122] [Fig 7] represents a variant of the solid phase extraction process,

[0123] [Fig 8] represents a variant of extraction device,

[0124] [Fig 9] represents a variant of the solid phase extraction process,

[0125] [Fig 10] represents a variant of extraction device,

[0126] [Fig 11] represents a variant of extraction device,

[0127] [Fig 12] represents the spectrum of N-glycans obtained from human plasma from an elution fraction at the outlet of the conduit after implementation of the method according to the invention for mass to charge ratios (m / z) of the identified molecules between 1000 and 5000 Da,

[0128] [Fig 13] represents the spectrum of N-glycans in Figure 6 for m / z ratios between 1100 and 2000 Da,

[0129] [Fig 14] represents the spectrum of N-glycans from Figure 6 for m / z ratios between 2000 and 3500 Da,

[0130] [Fig 15] represents the spectrum of N-glycans from Figure 6 for m / z ratios between 3400 and 4500 Da, and

[0131] [Fig 16] represents two spectra obtained for the same extraction process according to the invention on the same sample before and after recycling of the porous monolith.

[0132] Detailed description

[0133] Figure 1 illustrates a device 10 for implementing an extraction method according to the invention. The device 10 comprises a porous monolith 20 integrated in a fluid conduit 30 so that any liquid passing through the fluid conduit from one side of the porous monolith 20 to the other passes through the porous monolith 20, the latter acting as a filter in the conduit 30.

[0134] The conduit is connected at one of its ends to a liquid supply member 40 of the conduit which can be removably connected to a liquid reservoir 50. The supply member 40 can also be connected to a pressure controller for controlling the pressure in the fluid conduit and for controlling the fluid circulation in the latter. The other end of the conduit can be free. The invention is however not limited to a free end. The latter could be connected to a needle-type liquid distribution member. Such a free end makes it possible to deposit the circulating solution at the outlet on an extraction support 60, in particular in a container, an analysis well, or a MALDI plate, in particular in view of its analysis by an ancillary device such as a MALDI-TOF, or in a container 62, in particular a disposal container for recovering the various solutions to be eliminated during the process.

[0135] The porous monolith 20 and the conduit 30 are cylindrical in the illustrated embodiment. However, it could be otherwise. The invention is not limited to a particular shape even if the cylindrical shape is preferred.

[0136] The porous monolith 20 has hierarchical porosity having macropores and mesopores, and has a diameter less than or equal to 3 mm, here substantially equal to 1 mm.

[0137] Figure 4 illustrates the different stages of an example of a porous monolith manufacturing process.

[0138] The method comprises a first step, not illustrated, of forming an aqueous solution of a pore-forming agent and a sol-gel precursor and optional additives, in particular an acid and / or a matrix-dissolving agent.

[0139] The pore-forming agent may be chosen from water-soluble polymers, in particular polyethylene glycol (PEG), poly(acrylic acid), sodium poly(styrene sulfonate), poly(ethylene imine).

[0140] The water-soluble polymer(s) may have a molecular weight of between 1,000 and 100,000 Dalton, preferably between 5,000 and 50,000 Dalton, even better between 5,000 and 30,000 Dalton.

[0141] The concentration of pore-forming agent, in particular PEG, may be between 0.015 g and 0.35 g per mL of sol, preferably between 0.02 and 0.2 g per mL of sol. These values ​​are related to the concentration of sol-gel precursor, in particular tetramethoxysilanes (TMOS), according to values ​​of 0.03 to 1 g of pore-forming agent, in particular PEG per mL of sol-gel precursor, in particular tetramethoxysilanes (TMOS), preferably according to values ​​of 0.06 to 0.6 g of pore-forming agent, in particular PEG per mL of sol-gel precursor, in particular tetramethoxysilanes (TMOS). It is chosen according to the size of the macropores desired for the final porous monolith.The sol-gel precursor may be chosen from alkoxides, in particular hydrolyzable and condensable organometallics, in particular zirconium alkoxides, in particular zirconium butoxide (TBOZ), zirconium propoxide (TPOZ), titanium, niobium, vanadium, yttrium, cerium, aluminum or silicon alkoxides, in particular tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), tetrapropoxysilane (TPOS), tetrabutoxysilane (TBOS), trimethoxysilane, in particular methyltrimethoxysilane (MTMOS), propyltrimethoxysilane (PTMOS) and ethyltrimethoxysilane (ETMOS), triethoxysilanes, in particular methyltriethoxysilane (MTEOS), ethyltriethoxysilane (ETEOS), propyltriethoxysilane (PTEOS), aminopropyltriethoxy silane (APTES) and mixtures thereof, e.g. TMOS.It is also possible to use precursors such as sodium silicates or titanium colloids, especially if the purity requirements allow it, i.e. are not too high.

[0142] The proportion of pore-forming agent in the sol and the proportion of sol-gel precursor in the sol are predetermined based on the characteristics, including total porosity and average macropore size, of a sample of known sol-gel matrices taken just after gelation.

[0143] The solution is then stirred for a predetermined time of between 5 min and 3 h, even better between 15 min and 2 h, at a substantially constant controlled temperature of between 0°C and 90°C, better between 0°C and 50°C. This stirring step makes it possible to initiate the sol-gel process to form a sol 5 before phase separation.

[0144] The soil 5 is then added in step 2 to a container 12 to at least partially fill said container 12 and at least one mold 15 contained in the enclosure 12.

[0145] The mold 15 can be positioned in the enclosure which is gradually filled with the soil 5 in such a way that the mold 15 fills gradually without the presence of air bubbles or chemical composition gradients. Filling can be done until the mold 15 is completely immersed. Partial immersion is also possible. Adding the mold to the soil 5 contained in the enclosure 12 is also possible.

[0146] The enclosure 12 may be configured to contain a plurality of molds 15, which may or may not be identical. The enclosure 12 may be cylindrical as illustrated or have any other shape. The enclosure 12 may be made of plastic, in particular PTFE, PP, PE, PC, PET, PVC, or glass or stainless steel. The mold(s) 15 comprise two openings 17 and 18 on opposite surfaces of the mold 15, at least one of the two openings 17 extending below the ground level after filling. Such openings allow the filling of the mold(s) 15 by filling the enclosure 12 containing the mold(s) 15 or by at least partial immersion of the mold(s) 15 in the soil 5 contained in the enclosure 12 and the circulation of the soil 5 between the interior and exterior of the mold(s) before total condensation of the latter.In the example illustrated, the mold(s) 15 are in the form of tubes open at both ends and extend vertically in the enclosure 12, but it could be completely different, the tube could be oriented differently in the enclosure and / or the mold could have another shape.

[0147] The mold(s) 15 may be entirely contained within the enclosure 12, as illustrated, or protrude from the latter. In the first case, the mold(s) 15 may be entirely or partially immersed in the soil 5 after filling.

[0148] The mold(s) 15 may be made of plastic, in particular PTFE, PEEK, FEP, PE, PP, or polylactic acid, or of glass or stainless steel, in particular fused silica or borosilicate.

[0149] The mold(s) may be made of a porous body.

[0150] The mold(s) can be formed by 3D printing or casting.

[0151] The largest transverse dimension of the cavity of the mold(s) 15, in particular the diameter d of this cavity, may be between 13 mm and 0.025 mm.

[0152] Once the sol 5 has been introduced into the enclosure 12 and the mold(s) 15, condensation is carried out in step 3 throughout the enclosure and the mold. This sol-gel transition may be followed by at least partial maturation (or aging) of the assembly. This step ensures the formation of homogeneous macropores of a similar nature in the formed sol-gel matrix 22, regardless of its shape and size.

[0153] During condensation, the temperature can be kept substantially constant, in particular between 15° and 90°C, preferably 25 and 70°C, for a period of between 10 min and 4 h. The duration of condensation and the predetermined temperature depend on the internal structure of the desired sol-gel matrix and the duration of stirring of the initial solution in the sol formation step. At least partial aging can last between 30 min and 2 weeks, in particular less than 72 h at room temperature. Preferably, the aging time is sufficiently short to avoid the formation of mesopores and / or micropores.

[0154] A block 22 of sol-gel matrix containing the mold 15 is then extracted from the enclosure 12 in step 4. In the case where the mold 15 is only partially immersed, this step may be optional as we will see later.

[0155] The mold 15 with the sol-gel matrix 25 that it contains is then extracted from the porous solid in step 5, for example by cutting the sol-gel matrix of the block 22 flush with the mold and then removing the mold 15 with the sol-gel matrix 25 that it contains, or by breaking the sol-gel matrix of the block 22 around the mold 15. In the case where the immersion was partial, it is possible to directly remove the mold 15 with the sol-gel matrix 25 that it contains from the block previously extracted or directly from the enclosure 12.

[0156] Then, the sol-gel matrix 25 is extracted from the mold 15 in step 6. This is achieved by means of controlled pressure exerted on the sol-gel matrix 25 while holding the mold 15. The pressure can be obtained either with a solid made of plastic, glass, such as a fused silica capillary for example, or any other material that is fairly robust and smaller than the mold 15, or with a gas at a controlled flow rate. The extraction operation can be facilitated by immersing the mold 15 and sol-gel matrix 25 assembly in a liquid. It is optionally possible to generate a slight pressure difference by gently tapping the mold 15 and sol-gel matrix 25 assembly to extract the sol-gel matrix 25.

[0157] Once the sol-gel matrix 25 has been extracted from the mold 15, the method may include a step of controlled generation of mesoporosity. This step may be carried out by immersing the sol-gel matrix 25 or the mold-sol-gel matrix assembly in a basic solution, for example an IM ammonium hydroxide solution, or by heating the material in water in the presence of a precursor, for example urea to generate ammonia in situ. Note that in the second method, it is possible to add ammonium hydroxide. This operation may last between 0.5 h and 50 h at a substantially constant predetermined temperature of the sol-gel matrix between 30°C and 150°C. This step may be carried out on several sol-gel matrices simultaneously, i.e. in the same bath, whether from the same block or not. Preferably, the pore size obtained is less than or equal to 50 nm, better between 2 and 50 nm.

[0158] The resulting sol-gel matrix is ​​then dried. To do this, it is placed in a closed container, in particular an autoclave, to be dried in critical or supercritical conditions, in particular under a flow of air or inert gas, in particular nitrogen (N2) for a period of between 10 and 20 hours. They are then subjected to a ramp of 0.5°C / min up to 350°C with a plateau of a few hours at this last temperature and under a flow of inert gas (other gases can be used).

[0159] This results in a self-supporting monolith ready for use.

[0160] The porous monolith obtained preferably comprises macropores, i.e. having a chosen dimension greater than or equal to 50 nm, and mesopores, i.e. having a chosen dimension between 2 and 50 nm.

[0161] H is preferably of substantially homogeneous structure throughout its volume, as can be seen in Figure 3.

[0162] The porous monolith(s) may have an aspect ratio, defined as its height over its largest transverse dimension, of between 0.2 and 100.

[0163] The process may involve post-manufacturing modifications to the porous monolith, including functionalization of the internal surfaces of the porous monolith. Functionalization may be carried out using liquid-phase or gas-phase processes, using organosilanes, including chlorosilanes (e.g. octadecyltrichlorosilane) and alkoxysilanes (octadecyltriethoxysilane, aminopropyltriethoxysilane, propyltrimethoxysilane), or even hexadimethylsilazane.

[0164] Alternatively, the mold(s) may have only one opening. This opens into the ground after filling to allow the circulation of soil between the mold and the enclosure.

[0165] Alternatively, the initial solution may be an emulsion or a templating solution containing sol-gel precursors.

[0166] The porous monolith 20 obtained by this treatment is self-supporting and can be integrated into the heat-shrinkable conduit 30.

[0167] The fluid conduit is semi-rigid and heat-shrinkable. It has, before shrinkage, an internal diameter greater than the external diameter of the porous monolith and after shrinkage an internal diameter less than the external diameter of the porous monolith. Preferably, the minimum diameter of the conduit is strictly less than the diameter of the porous monolith. It can be between 95% and 85% of the diameter of the porous monolith.

[0168] The porous monolith is integrated into the conduit by insertion into the conduit before shrinking and then heating the conduit to a temperature depending on the heat-shrinkable material, in particular greater than or equal to 70°C, so that the conduit shrinks onto the porous monolith without damaging it until its diameter shrinks such that the porous monolith is fixedly encapsulated and any liquid solution passing through the conduit passes through the porous monolith, i.e. without a space between the internal wall of the conduit and the external wall of the porous monolith, as can be seen in Figures 2 and 3.

[0169] It is also possible to integrate several porous monoliths in parallel to process several samples or several fractions of a sample.

[0170] We will now describe a method for solid phase extraction (SPE) of compounds of interest from a sample.

[0171] The process involves passing different successive solutions through the porous monolith to extract the compounds of interest. All of the successive solutions can be inserted beforehand at the head of the conduit, i.e. upstream of the porous monolith.

[0172] The extraction method may first comprise a conditioning step by passing a conditioning solution 200 through the porous monolith 20. It makes it possible to prepare the porous monolith for the adsorption of the compounds of interest by impregnating it with a solution of polarity close to that of the sample. The conditioning solution 200 may be an aqueous solution of acetonitrile, for example 80% by mass in acetonitrile. The conditioning solution is eliminated at the outlet.

[0173] The sample 300 is then passed through the porous monolith 20. During its passage, the compounds of interest present in the latter adsorb to the surfaces of the porous monolith due to their greater affinity for the surfaces than for the solvent of the sample. The sample 300 can be passed once through the porous monolith. Alternatively, it is possible to pass it several times by making it go back and forth through the porous monolith 20. The liquid of the sample remaining at the outlet can be removed. The porous monolith 20 is then washed with a washing solution 400 for which the compounds of interest have a lower affinity than for the walls of the porous monolith 20. The washing solution 400 can be a 95% by mass aqueous acetonitrile solution. The washing solution 400 is removed at the outlet of the porous monolith.

[0174] Finally, as illustrated in Figure 6, a succession of successive eluent solution fractions 500 are entrained through the porous monolith. Each eluent solution fraction is recovered at the outlet of the porous monolith on different spots or wells 520 of an analysis plate. The eluent solution is a solution for which the compounds of interest have more affinities, in particular pure water, so that the compounds of interest are entrained in the eluent solution fractions. The different eluent solution fractions are recovered at the outlet for analysis. Analysis of the composition of the different eluent solution fractions makes it possible to deduce the proportion of compounds of interest in the initial sample. The analysis is carried out for example by MALDI-TOF, LC-FLUO or CE-LIF, in particular in the case of N-glycans.

[0175] In a variant illustrated in FIG. 7, the porous monolith 20 is encapsulated in a heat-shrinkable conduit 30 of non-cylindrical shape, in particular of conical shape.

[0176] The passage of the different solutions, in particular the conditioning solution 200, the sample 300, the washing solution 400 and the eluent solution 500 can be done through the porous monolith by one or more suction then discharge steps, as illustrated by the double arrow at each step in Figure 7. The different solutions therefore pass at least twice through the porous monolith 20 in one direction then in the other. After passing through the porous monolith, the solutions to be eliminated are recovered in elimination containers 62 and the solutions to be analyzed are recovered in an analysis container or well or deposited directly on a MALDI analysis plate 60.

[0177] In a variant illustrated in Figure 8, the fluid conduit 30 after encapsulation of the porous monolith is inserted into a solid phase extraction cartridge 80, in particular of the syringe body type. The different solutions are then recovered after passing through the porous monolith by the outlet nozzle 82 of the extraction cartridge 80. Such an extraction cartridge can be used as described previously or as described below. In a variant illustrated in Figure 9, the passage of the solutions 200, 300, 400 and 500 through the porous monolith 20 can be forced by centrifugation. The porous monolith 20 can be integrated into an extraction cartridge, the upper end of which can be closed by a plug 84. The conditioning solution 200 is added to the extraction cartridge 80 upstream of the porous monolith 20 and the cartridge 80 is closed using the plug 84. The cartridge is arranged above a disposal container 62.The assembly formed by the container 62 and the cartridge 80 is centrifuged so as to pass the conditioning solution through the porous monolith 20 and recover it in the container 62. The sample 300 and the washing solution are passed through the porous monolith 20 and recovered in the same container or another container 62 in the same manner. The cartridge is then inserted into an extraction container 60 to recover the eluate by passing the eluent solution 500 through the porous monolith in the same manner.

[0178] In a variant illustrated in Figure 10, all the steps of the method can be carried out using a multi-well system 900 comprising a vacuum base 910, receiving a container or a multi-well plate 60, on which an extraction plate 920 is mounted. The extraction plate 920 comprises a matrix of extraction cartridges 80 in which the porous monoliths are integrated. This plate 920 is placed, depending on the step of the method, on a removal container 62 not illustrated to recover the solutions to be eliminated or on a multi-well plate 60 having an extraction well 520 per extraction cartridge to recover the eluate at the outlet of each cartridge. The passage of the different solutions through the porous monolith can be forced using an air suction pump in the vacuum base 910. Such a system makes it possible to carry out several extractions simultaneously.

[0179] In the variant illustrated in Figure 11, the extraction can be carried out by solid phase extraction. In this case, the porous monolith can be integrated into a fluid conduit closed at its end, here a container, in particular of the Eppendorf® tube type. The porous monolith can extend to the bottom of the container and be fixed to the bottom of the container. The different solutions are then added to the container containing the porous monolith. The tube is shaken to accelerate the exchanges between the solutions and the porous monolith. The solutions are then extracted from the container, in particular by turning the latter over or by pipetting. Below is detailed the N-glycomic analysis of a human plasma sample.

[0180] A self-supporting monolith of about 800 pm diameter, having macropores of about 2 pm and mesopores of about 15 nm generated by immersion in a basic solution is fabricated.

[0181] A solution is prepared by mixing 0.33 g of PEG with 2 mL of TMOS in 4 mL of 0.01 M acetic acid. The solution is stirred at 0°C for 30 min to form a sol and then transferred into a polypropylene (PP) container in which a PTFE tube of approximately 1 mm diameter has been previously positioned vertically. Filling is carried out by gradually adding the sol into the enclosure from the lowest point using a micropipette. The amount of solution added is such that the mold is completely submerged.

[0182] The chamber is placed at a temperature of 40°C, and gelation occurs between 45 and 50 min after transfer to the chamber. After gelation has taken place, the gel is left to age for 24 hours at 40°C. Then, the sol-gel matrix resulting from gelation and maturation is extracted from the chamber and broken with metal pliers to recover the mold that has been incorporated into it. The monolithic sol-gel matrix encapsulated in the mold is then extracted using manual pressure exerted by a tube with a diameter of less than 1 mm. For this protocol, this pressure by a solid tube is sufficient to achieve the extraction of the monolith and does not weaken the gel.

[0183] The sol-gel matrix obtained is quickly immersed in a solution of IM NH4OH, respecting a ratio of approximately 5 between the volumes of basic solution and the volume occupied by the sol-gel matrix.

[0184] The resulting matrix is ​​then placed in an autoclave. The latter is placed in an oven and connected by tubes that allow gas circulation. The gel is then dried for 12 hours under N2. Finally, a heat treatment is carried out with a ramp of 0.5°C / min up to 350°C and a 2-hour hold at this last temperature.

[0185] The 0.8 mm diameter monolith is placed in a PTFE heat-shrinkable tube with an internal diameter before shrinkage of 1.27 mm. The tube is at least 10 cm long.

[0186] The assembly is then subjected to a localized temperature increase at the location where the monolith is positioned, using a heating gun set to a temperature of 500°C. The heat is manually distributed over the tube by regularly moving the gun nozzle and shrinkage occurs gradually to encapsulate the monolith. The shrinkage of the tube is visually checked. The diameter of the PTFE after shrinkage is less than 0.7 mm. This step can also be carried out in an oven at a temperature of 350°C for at least 10 min.

[0187] The tube containing the encapsulated porous monolith is ready for use. It is then connected to a pressure controller via a reservoir connector in which four containers can be placed, one for each solution (conditioning / sample / washing / elution).

[0188] At the outlet of the device, the fluids introduced into a waste container are recovered (the first three stages) and the eluates (last stage) are deposited directly onto a MALDI plate.

[0189] 34 pL of diluted plasma (containing 5 pL of collected plasma) is added to 100 mM sodium phosphate buffer pH 7.4 (10 pL) and 10 mM dithiothreitol (5 pL) before denaturing plasma glycoproteins by heating at 95 °C for 5 minutes. After cooling to room temperature, 2 pL of PNGase F solution (1 U / pL) is added and protein deglycosylation is carried out overnight (approximately 16 hours) at 37 °C. Residual glycosylamines are converted to glycans by incubation with 5 pL of 1 mol / L hydrochloric acid for 45 minutes at 37 °C.

[0190] A 0.25 M EDC / 0.25 M HOBt mixture is prepared in ethanol as an activating agent to carry out ethyl esterification reactions, which induce the esterification of α2,3-linked sialic acids and the lactonization of α2,6-linked sialic acids.

[0191] The derivatization is performed by adding 3 pL of previously released N-glycan sample (equivalent to 0.3 pL of human plasma respectively) to 20 pL of esterification reagent respectively, followed by incubation with shaking at 350 rpm for 1h30 at 37 °C. After that, 50 pL of 80% ACN is added to form the extraction sample, 10 minutes before proceeding to the extraction of N-glycans with the silica monolith and MALDI-TOF-MS analysis.

[0192] For each step, the solutions are loaded into a container and directed by positive pressure using a pressure controller into the PTFE tube containing the material. Negative pressure can also be used with the pressure controller. Positive and negative pressures can also be applied with syringe pumps or even manually. This device can also be used to perform suction-discharge cycles to achieve purification.

[0193] The PTFE-encapsulated monolith is conditioned and equilibrated with 3 x 1 mL of pure water followed by 3 x 1 mL of 80% aqueous ACN. Then, the 73 pL of the extraction sample is loaded, then washed with 20 pL of 95% ACN. Finally, the N-glycans are eluted in 10 successive fractions of 1 pL of pure water. Each fraction is deposited directly using the device onto the MALDI plate. Note that in the protocol, no trifluoroacetic acid is used.

[0194] 1 pL of the 2,5-DHB matrix solution (2,5-dihydroxybenzoic acid, 10 mg / mL in 50% methanol) is added and mixed with each of the 1 pL fractions deposited during direct elution on the plate. After an initial crystallization, 0.2 pL of ethanol is deposited to recrystallize and improve the reproducibility results during the analyses.

[0195] Finally, each spot on the MALDI plate is analyzed with a MALDI-TOF / TOF instrument, including Bruker's UltrafleXtreme equipped with a laser, including Bruker's Smartbeam-II. Regarding acquisition conditions, mass spectrometry spectra were acquired at a laser repetition rate of 2 kHz in positive mode, with an accelerating voltage of 20 kV and an extraction delay of 130 ns. Spectra are obtained by accumulating 5,000 shots over mass windows of 1100 to 5000 Da for plasma N-glycans, and 500 to 5000 Da for human milk free oligosaccharide samples (HMO, see Example 2).

[0196] After implementing the purification protocol for human plasma, N-glycan profiles on fractions 2 to 4 were obtained by mass spectrometry with an optimal spectrum obtained for the third fraction.

[0197] A calibration was performed and the peaks identified by the mass spectrometry device software.

[0198] The spectrum obtained is illustrated in Figures 12 to 15. The analysis made it possible to differentiate more than 58 plasma N-glycans of different compositions, some of which are illustrated, which correspond to more than 81 species of N-glycans due to the variation of the sialic acid linkage. The 8 majority structures were found, namely H5N4E2 (m / z 2301), H5N4E1 (m / z 1982), H5N4E1L1 (m / z 2255), H6N5E2L1 (m / z 2940), H5N4F1E1 (m / z 2128), H5N4F1E2 (m / z 2447), H6N5F1E2L1 (m / z 3086) or H6N5E2L1 (m / z 2986). The proportions of mono- and bis-sialylated biantennary glycans, H5N4E1 (m / z 1982) and H5N4E2 (m / z 2301), which are the two major structures usually found, present proportions of 1:3.

[0199] Example 2

[0200] A cylindrical self-supporting porous monolith with a diameter of 5 mm and a length of 9 mm was fabricated by the same method as described in Example 1. It was embedded in a PTFE heat-shrinkable tube with a length greater than 9 mm by the embedding method of Example 1 and then placed in an SPE-type cartridge, as illustrated in Figure 8.

[0201] The SPE cartridge containing the encapsulated porous monolith is ready to be used for extraction. The SPE cartridge is placed under vacuum at -0.2 bar.

[0202] 50 μL of human milk was diluted, vortexed, and then centrifuged at 15 kG at 2°C for 30 minutes. After this, the supernatants were removed and 200 μL of acetonitrile (ACN) was added to the samples before vortexing.

[0203] The SPE cartridge containing the monoliths was conditioned and equilibrated successively three times with 1 mL of water, then three times with 1 mL of 80% ACN. Then, the samples were loaded and washed 12 times with 200 pL of 95% ACN. Finally, the elution of free milk oligosaccharides (HMO) was carried out with 200 pL of FLO in a 2 mL tube before being dried under a nitrogen flow and analyzed by mass spectrometry. The spectrum obtained is illustrated in Figure 16, spectrum a.

[0204] After this first extraction, the cartridge is removed. The porous monolith and the PTFE are heat-treated under nitrogen flow at an initial temperature of 40°C followed by a 210-minute ramp up to a final temperature of 250°C for two hours. Then, the treated part is recovered and replaced in the cartridge and the previous extraction protocol is used again. The conditioning and elution fractions were recovered and analyzed by mass spectrometry.

[0205] In the conditioning fractions of this second extraction, no oligosaccharides or other residual compounds were detected, indicating the good regeneration of the porous monolith. Regarding the eludon fractions, free oligosaccharide profiles were obtained. An example of the mass spectrum obtained is illustrated in Figure 16, spectrum b. It can be seen that the spectra in Figure 16 obtained respectively by the first and second extractions are similar, which clearly indicates that the porous monolith can be used several times.

[0206] The invention is not limited to the examples just described. For example, the porous monolith has a shape other than those described. The fluidic device may be other than as described, in particular the pressure control may be done differently.

Claims

Demands 1. A solid-phase extraction process for one or more compounds of interest from a liquid sample comprising: the integration of a self-supporting porous monolith (20) into a fluidic conduit (30) such that the self-supporting porous monolith (20) is fixed in the fluidic conduit (30) during said process and forms a filter in the fluidic conduit, at least one passage of the sample (300) through the porous monolith in the fluidic conduit (30) over at least a portion of the porous monolith (20), the self-supporting porous monolith (20) having a largest transverse dimension to the fluidic conduit (20) less than or equal to 3 mm, the fluidic conduit having one or more open ends before integration, the end or at least one of the ends remaining open during the integration step of the porous monolith.

2. A method according to claim 1, wherein the self-supporting porous monolith (20) is formed by a manufacturing process comprising: the formation of a soil (5) comprising a sol-gel precursor in aqueous solution and, preferably, a porous agent; the at least partial filling of a chamber (12) and of at least one mold (15) contained within the chamber (12) with previously formed soil; the mold (15) comprising at least one opening (17) opening in the soil after filling with soil; the formation of a sol-gel matrix (22) within the chamber from the soil; the extraction of the mold (15) with the sol-gel matrix contained within the mold (25) from the chamber; and the extraction of the sol-gel matrix (25) from the mold (15); the formation of a porous monolith (20) from the sol-gel matrix extracted from the mold (25); and the formation of the soil, the sol-gel matrix, and the porous monolith. doing so by a sol-gel process.

3. Method according to claim 1 or 2, the porous monolith having hierarchical porosity, in particular comprising macropores, in particular macropores with a dimension greater than or equal to 50 nm, and mesopores, in particular with a dimension less than or equal to 50 nm, preferably between 2 and 50 nm.

4. A method according to any one of the preceding claims, wherein the porous monolith has a larger transverse dimension to the conduit less than or equal to 2 mm, better less than or equal to 1.5 mm, even better less than or equal to 1 mm.

5. A method according to any one of the preceding claims, comprising post-fabrication modifications of the porous monolith (20), in particular functionalization of the surface of the porous monolith, in particular before or after its integration into the fluidic conduit (30).

6. A method according to any one of the preceding claims, wherein the fluidic conduit is made of a heat-shrinkable polymer, in particular polyolefin, such as polyethylene (PE) or polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyetheretherketone (PEEK), fluorinated ethylene propylene (FEP) or polyvinylidene fluoride (PVDF) or a mixture thereof, and the integration of the porous monolith (20) into the fluidic conduit (30) comprises inserting the porous monolith (20) into the fluidic conduit (30) and heating the fluidic conduit (30) to shrink the conduit so as to encapsulate the porous monolith in said conduit, in particular for a duration and at a temperature chosen so that the fluidic conduit shrinks until it comes into contact with the outer wall of the porous monolith over at least part of the length of the porous monolith, preferably over the entire length of the porous monolith.

7. A method according to any one of the preceding claims, wherein the porous monolith retains its integrity during the integration step in the fluidic conduit.

8. A method according to any one of the preceding claims, wherein the heating of the conduit is done at a temperature greater than or equal to the minimum shrinkage temperature of the conduit and less than or equal to the melting or degradation temperature of the fluidic conduit.

9. A method according to any one of the preceding claims, wherein the fluidic conduit has, at its open end or one of its ends, after integration of the porous monolith, a non-zero length of conduit devoid of porous monolith.

10. A process according to any one of the preceding claims, the process being a solid-phase extraction process of compounds of interest by adsorption of these compounds onto the surfaces of porous monoliths to separate them from the rest of the sample, then recovery by elution of compounds of interest adsorbed on the surfaces of the porous monolith.

11. A method according to any one of the preceding claims, comprising, before passing the sample, conditioning the porous monolith by passing one or more successive conditioning solutions through the fluidic conduit via the porous monolith and / or, after passing the sample through the fluidic conduit, washing the porous monolith by passing one or more washing solutions through the fluidic conduit via the porous monolith.

12. A method according to any one of the preceding claims, comprising, after passing the sample and where appropriate after washing, eluting the compounds of interest by passing an eluent solution or several fractions of eluent solution through the porous monolith in the fluidic conduit and recovering the eluent solution or each fraction of eluent solution after passing through the porous monolith, in particular for analysis to determine its composition in compounds of interest, particularly in dissociated recovery systems, for example on different areas of a plate, in particular a mass spectrometer plate using a matrix-assisted laser desorption / ionization source coupled or not to a time-of-flight analyzer.

13. A method according to the preceding claim, comprising analysis by mass spectrometry, in particular a mass spectrometer coupling a matrix-assisted laser desorption / ionization (MALDI) type source to a time-of-flight (TOF) analyzer, by liquid chromatography coupled with fluorescence detection (LC-Fluo) or by capillary fluorescence-coupled electrophoresis (CE-LIF), after recovery of the eluent solution without a prior step of drying or concentrating the eluent solution.

14. A process according to any one of the preceding claims, comprising recycling the porous material by heat treatment.

15. A method according to any one of the preceding claims, wherein the sample (300) passing through the porous monolith (20) comprises free N-glycans or oligosaccharides, and the porous monolith is configured to adsorb these species contained in the sample as the latter passes through the porous monolith, preferably the method additionally comprising: washing the porous monolith (20) by passing a solution of washing (400) through the porous monolith, elution of N-glycans by passing an eluent solution (500) through the porous monolith, collection of the eluent solution at the outlet of the porous monolith, - glycomic analysis of the eluent solution collected.

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