• 专利附录
  • 专利说明
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    • 专利摘要:
    The present invention relates to a micro-structured liquid sample support substrate comprising a lower face and an upper face which comprises, on the one hand, at least one surface cavity of volume V0 opening on said upper face and forming an analysis zone. of the liquid sample and secondly, a first groove of a first volume V1 disposed around each surface cavity and opening on its upper face. According to the invention, this substrate is characterized in that it further comprises at least one second groove (14,15,16) opening on its upper face and arranged around the first groove (13), the sum of the volumes of the second grooves being equal to V2, the volume V1 + V2 being greater than or equal to 0.05 V0, preferably 0.1 V0. The invention also relates to an analysis assembly comprising the substrate and its use.
    公开号:FR3040895A1
    申请号:FR1501889
    申请日:2015-09-11
    公开日:2017-03-17
    发明作者:Mael Leberre；Adrien Plessis
    申请人:Elvesys；
    IPC主号:
    专利说明:

    BACKGROUND OF THE INVENTION The invention relates to a microstructured substrate for a liquid sample support comprising a lower face and an upper face which comprises, on the one hand, a surface cavity of VO volume opening on said upper face and forming an analysis zone of the liquid sample and secondly, a first groove of a first volume VI, disposed around said cavity and opening on its upper face.
    It also relates to a set of analysis of a liquid sample comprising such a substrate, such as a set of substrate, liquid sample and slide placed thereon, as well as a use of such a substrate for carrying out analysis. of liquid sample.
    In many areas of research and analysis, we seek to determine the presence or absence of certain particles called "particles of interest" in a liquid and / or to study the development of these particles of interest present in this liquid and / or to promote the formation of structures from the particles present in the liquid. These particles may be on the one hand biological objects such as cells (animal and / or plant, living and / or non-living), antibodies, proteins, viruses, etc ... and / or secondly non-biological objects such as molecules of interest, in particular chemical molecules, in particular polymers, liquids, etc.
    Most of the protocols implemented for the analysis and / or the study of a sample generally consisting of a liquid containing (or not) at least one particle, use a sample support which is frequently constituted of a parallelepipedal glass slide and an observation instrument such as a microscope, a spectrum analyzer, a fluorescence quantization system, etc. The sample is deposited on the glass slide, then a glass slide is gently deposited on the sample as shown in Fig.3 of this text, so as not to enclose an air bubble between the lower surface of the glass. lamella and the sample nor to damage the particles possibly present in the sample, the lamella then being maintained by capillarity on the glass slide. In some applications, it can be fixed on the glass slide by gluing, usually with a wax, in particular to enclose the sample and avoid the evaporation of the latter which could occur during observations continuous especially long and / or when the sample is subjected to temperature variations, including temperature rises.
    By limiting the evaporation of the liquid, the bonding technique makes it possible, on the one hand, to avoid or limit the movement of the liquid in the analysis zone of the sample and, on the other hand, to avoid a variation in the concentration of the liquid. particles and / or salts possibly dissolved in the sample, which allows observation of the particles under stabilized conditions.
    However, the use of this type of protocol creates many difficulties for the user. First, when depositing the liquid on the slide, the drop may spread uncontrollably over the entire surface in a film of a few microns thick. In addition to the loss of sample along the edges of the blade, it then becomes difficult to control the lateral extension of the liquid, for example relative to an analysis zone imposed by the analysis instrument. Other capillary phenomena can also disturb the positioning of the sample: during the removal of the upper lamella, it is common that the affinity of the liquid for one of the two substrates causes the drop to quickly migrate to the one of the edges of the blade and lamella. This phenomenon is also at the origin of the wasting of a part of the sample and makes the positioning of the sample in the center of the lamella more complex. The latter then takes a random lateral extension, and therefore has a thickness difficult to control. Finally, the presence of liquid at the edge of the coverslip can disrupt the bonding step that makes the system hermetic. Since this step depends on the dexterity of the user, the result of the conditioning of the sample in the form of a thin cavity is very random and this can result in a considerable variability of the analysis results. The presence of liquid outside the blade and / or the coverslip, which can thus be in contact with the user and / or the analysis instrument, is generally considered unacceptable particularly in the case of samples containing carcinogenic or toxic products.
    Finally, the gluing step is particularly lengthy to implement and requires the intervention of an experienced user. For this reason, this step is not possible for certain applications that require the analysis of a large number of samples. In addition, it does not allow the user to recover his sample since this type of bonding is not suitable for a reversible opening.
    In an attempt to overcome some of these disadvantages, various solutions have already been proposed: US-A-2041290 discloses a sample holder in the form of a wafer for the analysis of a liquid sample, for example a droplet of blood, on the upper surface of which are provided a plurality of wells of circular shape, each well being surrounded by a channel provided to collect the excess liquid of the sample to be deposited deposited in said well. US-A-2302830 discloses a wafer of the same type in which the well is formed by depositing a circular bead of material on the surface of the wafer, this bead being surrounded by a circular groove etched on the surface of the wafer to collect the excess of liquid to be analyzed. US-A-5948685 proposes to use a device consisting of a microscope glass slide on which is structured a restraining barrier whose thickness is less than 1 μm, so as to create a well on the surface of the blade . The sample being placed inside this well, any overflow is in principle stopped, which makes it possible to avoid the effects of loss of liquid at the edges of the lamella. However, it has been found that if the volume of the sample is poorly controlled, ie too high, this barrier is not sufficient to prevent leakage at the edges. In addition, if the slat that is just placed on the sample has a high affinity with the liquid sample, which is often the case and if it is not deposited strictly parallel to the blade, a phenomenon overflow of the liquid takes place along this coverslip. WO 82/02958 discloses a support wafer for the analysis of liquid samples, this wafer comprising a plurality of sets provided for depositing a drop of liquid sample, each set consisting of a central well surrounded by a channel or groove capable of receiving the excess liquid deposited in the central well. US-A-7138270 describes in particular in Fig.8 a liquid sample carrier having a plurality of circular wells evenly distributed on the upper surface of the support. The typical height of the side walls of the wells is between 30 and 200 μm, all of these wells being surrounded by an external confinement barrier. This type of configuration makes it possible to completely fill the wells with the aid of an excess of liquid which is removed during the placing of the upper lamella, in the intermediate zone situated between the different wells and delimited by part of the side walls of the various wells and secondly by the outer containment barrier. This external barrier makes it possible to contain the excess liquid to avoid any leakage outside the blade and the coverslip. In this type of system, the positioning of the excess liquid is however not controlled and it is necessary to deposit the coverslip in a suitable manner to limit cross contamination between the wells. Indeed, as the upper plate is not hermetically sealed to the well structures, some of the excess liquid can be drained by capillarity out of a well uncontrollably. This has the effect of generating a bubble within one or more wells (if the amount of liquid drained is too large) and the diffusion of particles from one well to another when the exhaust fluids of two wells consecutive events, thus contaminating the subsequent analysis of initially independent wells. In addition, the smallest dust or particle from the sample interposed between the barrier and the upper lamella or the slightest irregularity in the barrier is at the origin of a preferred non-controllable evaporation zone which limits the reproducibility the analysis, especially when the latter is long and requires to be performed at high temperatures. This configuration is therefore unreliable for routine analysis even if it is performed with particularly reproducible (and therefore expensive) technologies.
    In order to limit the problems of evaporation and leakage, it has been proposed in WO2006052492 to first deposit a layer of rough glue on the confinement barrier. The disadvantage of this solution is the impossibility of recovering the sample, the loss of performance of this type of device in case of dust or defect in the manufacturing process and especially the risk of diffusion of the glue compounds into the surface. sample to be analyzed which would then be contaminated.
    None of the aforementioned systems therefore makes it possible to provide a satisfactory solution to the problem of positioning and maintaining a liquid sample in a fine cavity, which allows precise lateral positioning of the sample, excellent fluidic and chemical stability of the sample in the measurement zone, as well as an absence of cross contamination in the case of the positioning of several samples on the support, regardless of the dexterity of the user and the precision of the volume of the sample.
    Furthermore, when the supports as described above are used with a sipe (usually transparent glass or any other similar material) which comes to rest on the liquid sample to trap it between the cavity and the lamella, so as to obtain an observable sample directly above the lamella, it is very common to note that a gaseous bubble has formed in the liquid at the cavity, under the lamella.The presence of this bubble which can move throughout the cavity makes the observation of the liquid sample difficult if not impossible especially when the area occupied by the bubble is large relative to the surface of the cavity. In an attempt to avoid the formation of this bubble due to a lack of liquid in the cavity, the experimenter tends to dispense a quantity of liquid significantly greater than the volume of the cavity. However, it was found that this did not prevent the formation of this bubble during the installation of the coverslip on a support as described above.
    An object of the invention is constituted by a substrate of the micro structured type which does not have the drawbacks of the supports of the prior art and makes it possible in particular to avoid the formation of a bubble in the liquid sample during the closure of the cavity of the support with a coverslip after having deposited the sample in the cavity.
    The microstructured liquid sample support substrate according to the invention which comprises a lower face and an upper face which comprises, on the one hand, at least one surface cavity of volume VO opening on said upper face and forming a zone of analysis of the liquid sample and secondly, a first groove of a first volume VI, arranged around each surface cavity and opening on said upper face is characterized in that it further comprises at least one second groove opening on its upper face and arranged around the first groove, the sum of the volumes of the second grooves being equal to V2, the volume VI + V2 being greater than or equal to 0.05 VO, preferably 0.1 VO.
    According to a first variant, the volume V1 + V2 is greater than or equal to 0.2 V0, more preferably greater than or equal to 0.3 V0.
    According to another variant, the substrate according to the invention is characterized in that it comprises in total n grooves, with n> 2, each groove of rank p greater than or equal to 2 and less than or equal to n being arranged around the row groove (p-1), the total volume V3 of the (n-1) first grooves being greater than or equal to 0.05 V0.
    According to a preferred embodiment, the substrate according to the invention will have n grooves, n being less than or equal to 100, preferably less than or equal to 10.
    According to a first alternative, the cross section of each of the grooves will be substantially identical and / or preferably constant or substantially constant: the substrate will comprise, for example, concentric circular grooves of the same section, preferably spaced equidistant from one another.
    According to another alternative, the section of at least one of the grooves, preferably the nth or last groove (the outermost) will be greater than that of the other grooves, preferably greater than or equal to 5 times, more preferably 10 times the section of the (n-1) th groove. The additional advantage of an nth groove of a volume substantially greater or much greater than that of the (nl) th groove, is to avoid in the vast majority of cases, an overflow of the sample liquid beyond this nth groove, in particular when a support having a plurality of cavities is provided, each of which is surrounded by a plurality of grooves, for example the same number n of grooves.
    According to a preferred embodiment, the substrate according to the invention has at least six, preferably at least eight surface cavities, each cavity having a diameter of about 5 mm and a depth of between 20 micron and 500 microns and being distant. about 5mm from another cavity, each cavity being surrounded by a series of 5 to 10 grooves of about 100 microns in depth, spaced from each other by about 100 microns.
    Preferably also the grooves are continuous and / or they do not communicate (fluidly) with each other. In general, it has indeed been found that the presence of at least one second groove disposed around the first groove makes it possible to ensure a uniformly distributed flow (over 360 degrees) of the liquid from the cavity to at least the second groove thus avoiding the "wedge effect" present when closing the cavity with a coverslip. By thus avoiding a sudden and therefore uncontrolled suction effect of the liquid, which causes too much flow of liquid, it avoids too much empty the cavity and cause the appearance of an air bubble under the coverslip.
    Preferably the distance between the first groove and the second groove will be substantially constant (for example in the case of a first circular groove, the second groove will preferably also be circular, the two grooves being concentric). This further improves the results obtained.
    Preferably also, these grooves without necessarily having an exactly constant cross-section on their whole, will generally have a substantially constant section so as not to create and / or favor privileged passages for the liquid during the flow thereof. cavity to the first groove and the first groove to the second groove, promoting symmetry of the cavity and groove assembly.
    The shape of the cavity, in particular the shape of the contour of the cavity when it opens on the upper surface of the support, may be arbitrary, but will preferably be in the form of a bowl with walls preferably substantially vertical or inclined towards the inside the cavity and whose contour will preferably be substantially symmetrical with respect to an axis perpendicular to the upper face of the support (generally flat). The preferred outline will be circular, rectangular or square. Preferably at least the first groove will be of the same shape as the contour of the cavity, positioned substantially equidistant from it.
    According to another variant, the surface situated on the upper face of the substrate, between the contour of the cavity and the inner contour of the first groove will preferably be a flat or substantially planar surface, preferably oriented parallel to the lower face of the support, it itself generally flat. This surface (hereinafter referred to as the first surface) may either retain its surface properties (for example hydrophilic or hydrophobic) inherent to the shape initially chosen for this surface and / or to the material used for the substrate or its top layer (see FIG. after, for example, the case of the substrate whose cavity and barriers are created by selective deposition of a layer of material), or be specially treated to give it surface properties different from those of the material used (for example, make it hydrophilic if it is hydrophobic or vice versa, or reduce and / or increase its hydrophilic or hydrophobic character).
    The treatments to be applied to a surface in order to modify its surface characteristics with respect to the different liquids present in the samples to be analyzed (whether these treatments are of a physical and / or chemical nature) are generally known to the human being. job.
    Of course and according to yet another variant of the invention, it may also be necessary to modify the surface state of one and / or more of the walls and / or wall portions of one or more grooves. similarly to that described above concerning the surfaces between the grooves (including the central cavity) in order to modify if this is desirable, the surface condition of at least a portion of said surfaces. It is also possible to modify the shape of any surface or surface portion of the substrate intended in particular to be in contact with the liquid sample in order to modify its capillary properties, in particular the contact angle of the liquid with said surface. It is also possible according to another variant of the invention, to modify the shape of the walls of the grooves and the central cavity, in particular at the edges present at the intersection of the upper face of the substrate and the side walls of the grooves or of the central cavity so as to create additional capillary barriers, the effect of which will be added (in more or less) to the capillary barrier effect inherent to the initial shape of the surface and / or the material of which is made this surface, with respect to the liquid of the sample. Throughout the present description, the term "capillary barrier" will be used to refer to any deformation of the surface or of a surface portion of the substrate intended to be in contact with the liquid of a sample, that this deformation results from a treatment of the surface and / or a modification of the shape of the surface, in particular the appearance of convex shapes on it.
    Thus, according to a variant of the invention, the support is characterized in that at least certain areas of the upper face and / or of the cavity and / or grooves are treated so as to modify in these zones the properties of the capillary barrier. inherent to the surface condition of the support material and / or the geometry of the grooves and surfaces.
    The section of the grooves can be any but will be in different preferred variants preferably rectangular, square, trapezoidal, triangular, etc.
    In particular, the shape of the section of a groove can be chosen as a function of the value of the angle (convex) that the side wall of the groove makes and the surface of the upper face at the level of the substrate (value of the angle convex of the edge) so as to modify the capillary barrier created by this convex surface which connects the grooves (or the cavity) to the upper face of the substrate.
    The zone of convexity at each capillary barrier and in particular each edge is characterized by an angle of convexity Alpha c defined as the angle between the local direction of the surface after the convexity zone (for example the lateral wall of a groove) and the overall direction of the surface (for example the direction of the upper face of the substrate). It has thus been found according to another variant of the invention that the capillary barrier thus created was particularly effective if Alpha c were such that:
    Alpha c> 140 ° - (Alpha m support + Alpha m surface) and preferably,
    Alpha c> 180 ° - (Alpha m support + Alpha m surface) - Alpha m support designating the wetting angle of the sample liquid on the surface of the lamella that is just deposited on the liquid sample disposed in the cavity of surface of the support according to the invention (also called blade). - Alpha m surface designating the wetting angle of the sample liquid on the convex surface containing the capillary barrier.
    Such a capillary barrier may take for example one of the following forms: A groove etched in the glass by hydrofluoric acid lithography having vertical flanks (thus forming a convex zone at its inner edge having a convex angle 90 °), then treat with a hydrophobic surface treatment making the wetting angle of the water on the glass> 70 ° so that the angle of the surface on the inner side of the groove. In this case, the support surface may be a clean glass slide (wetting angle of about 20 ° for pure water and clean glass). a resin structure of the "novolac" epoxy type sold under the name "SU8" in the form of a wall defined by photolithography on a glass slide, and having vertical flanks. This type of surface can also form a capillary barrier with a clean glass slide (SU8 resin at a wetting angle of about 70 °). - A rounded valley molded in a polyolefin such as polyethylene whose inner side forms an angle (Alpha c) of 70 ° with the surface (thus easy to mold or form by embossing, for example). This type of surface can also form a capillary barrier with a clean glass slide (the PE at a wetting angle of 96 °). Grooves etched by micromachining in a surface of polytetrafluoroethylene (PTFE) or its derivatives or neighboring fluorinated compounds having similar properties, with flanks having a convexity angle of 135 °. This type of surface can form a capillary barrier for any surface with water, but also for many other liquids. Advantageously, the convex zone may contain an edge which defines the angle of convexity accurately and well localized. Advantageously, the surface after the convex zone may be locally more hydrophobic (less wetting) to increase the efficiency of the capillary barrier while retaining a freedom on the choice of the nature of the surface of the remainder of the surface, in particular the sample cavity (Surface treatments to promote adhesion of adherent cells, for example, are often hydrophilic, so it may be necessary to keep a hydrophilic surface for this area). In general, the material of the support may not only be glass usually used for these analysis plates but may also be constituted by any thermoplastic material, thermosetting, transparent or not, as well as ceramic or other materials.
    The dimensions of the grooves may be very varied depending on the intended use of the support but are not critical for the implementation of the invention. Their depths may vary between about 1 nanometer and 500 microns, preferably between 20 microns and 500 microns.
    Preferably, the distance between the inner contour of a groove with the outer contour of the preceding groove that it surrounds and / or the cavity is between about 1 micron and 1 mm, preferably about 100 microns.
    According to a first variant, the substrate is constituted by a monolayer or multilayer material in which are engraved or thermally, mechanically and / or chemically formed at least one of the grooves and / or the cavity.
    According to a second variant, the substrate is composed of at least a first lower layer on which is deposited a second upper layer from which are formed the side walls of the cavity and / or grooves, the side walls of which are thus arranged in protrusion with respect to the lower layer. According to this variant, the grooves may be made by selective deposition of at least one resin or by vacuum deposition of PVD, CVD, PECVD type, etc.
    The local treatment of certain portions of grooves and / or cavities or the surface of the substrate to make these portions more or less hydrophilic or hydrophobic can be achieved by any means well known to those skilled in the art.
    The manufacture of the grooves and / or the cavity does not pose any particular problem: it is sufficient to use the well-known and identified forming methods for the material used: mechanical, thermomechanical, chemical etching (selective etching with the aid of a masking technique), laser engraving, as well as all the techniques for selective deposition of materials on a support when the cavity and / or the grooves are created by a deposition technique (coating, for example through a silk screen, chemical deposit, vacuum deposition type CVD, PVD, etc ..).
    Of course when the cavity and / or the grooves are created by depositing a material on a support, the cavity and / or the grooves are not created by removing material on and from the upper surface of the support but by contribution of a material identical to or different from that constituting the support to create physical barriers that delimit the cavity and / or the peripheral grooves. For reasons of simplification, the terms cavity and groove will be used to designate respectively the volume trapped within the first physical barrier (which is therefore continuous in this variant) and the volumes trapped between the other barriers arranged around the first barrier and therefore around the central cavity (this variant is illustrated in particular in the figures below)
    It has generally been found that with the microstructured support according to the invention, the spreading zone (3) of the liquid sample (1) respects the envelope delimited by the grooves, thus making it possible to center the sample in a selected and non-random area of analysis and also limiting the risk of loss of sample leakage.
    Among the preferred structures of the support according to the invention, there are the structures comprising two or three grooves arranged around the central cavity, these grooves being preferably similar, ie preferably having the same cross-section, as well as the structures in which last groove has a larger section than the other grooves. The invention also relates to a set of analysis of a liquid sample characterized in that it comprises a substrate (10) as defined above, on which at least one liquid sample (1) is deposited in at least one cavity (12) of the substrate (10) which is covered by a lamella (30) closing all the cavities (12) of the substrate (10), at least one of the intersections between the lateral surfaces of the grooves (13, 14,15,16) and / or the cavities (12) and the upper surface (17) of the substrate (10) being a surface or convex shaped surface portion whose angle of convexity Alpha c defined as the angle between the local direction of the lateral surfaces of the grooves (13, 14, 15, 16) and / or the cavities (12) and the direction of the upper surface (17) of the substrate being such that:
    Alpha c> 140 ° - (Alpha m support + Alpha m surface), preferably
    Alpha c> 180 ° - (Alpha m support + Alpha m surface) - Alpha m support designating the wetting angle of the sample liquid (1) on the surface of the strip (30) that is just deposited on the liquid sample (1) disposed in the surface cavity (12) of the support (10) - Alpha m surface designating the wetting angle of the sample liquid (1) on the surface or convex shaped surface portion which connects the grooves or cavities at the upper surface (17) of the substrate (10). In general, the grooves will be continuous and separated from each other. Although it is possible to communicate two or more wells and / or grooves between them and in particular several wells with each other and to provide a set of grooves surrounding the plurality of wells (as for example represented in FIG. 6 of the present application or the grooves are common to several cavities), it will be preferable not to provide liquid communication between the different grooves, between them and / or with at least one cavity.
    The substrate defined above as well as the analysis set find their use especially for carrying out analysis of a liquid sample and in particular using the amplification technique called "PCR" (or "Polymerase Chain Reaction") . The invention will be better understood with the aid of the following exemplary embodiments given in a nonlimiting manner together with the figures which represent: FIG. 1, a schematic view of a system consisting of the micro structured substrate according to the invention covered with a wafer during a microscopic observation type analysis. FIG. 2 is a schematic view of the filling steps of the micro-structured zone of the substrate according to the invention.
    Figure 3 is a schematic view of the steps of filling a substrate of the prior art.
    Figure 4, schematic examples of exhaust networks (grooves) of triangular and trapezoidal sections.
    Figure 5 is a schematic view of an example of a substrate according to the invention for multi-sample analysis.
    In all the figures, the same elements bear the same references.
    FIG. 1 is a diagrammatic cross-sectional view of an exemplary embodiment of the support according to the invention in end use on which is shown a schematic sectional view of the assembly according to the invention consisting of a micro structured substrate composed of a thick material having a lower face 11 and an upper face 17 on which opens a network of surface cavities. This network of surface cavities is composed of a first surface cavity 12 called "analysis zone" intended to receive the liquid sample (1) to be analyzed, above which is placed according to the present example a conventional microscopy system via a microscope objective (2). The network of surface cavities further comprises a plurality of grooves 13, 14, 15, 16 forming an area called "exhaust zone" intended to collect the excess sample (1) initially deposited by the user in the analysis zone and / or during the installation of the transparent plate 30 on the support at the level of the analysis zone. The exhaust zone consists of a set of grooves arranged concentrically preferably when the central cavity 12 has a center of symmetry.
    The grooves, hereinafter also called "exhaust passages" are in this example four in number and such that the groove 16 surrounds the groove 15 which surrounds the groove 14 which surrounds the groove 13 which itself surrounds the central cavity 12 or analysis area. The exhaust corridor 13 constitutes the exhaust corridor hereinafter called "primary", that is to say surrounding only the cavity or zone of analysis 12. (the other exhaust passages 14, 15, 16 are hereinafter called "secondary", that is to say surrounding the analysis zone 12 and the primary exhaust corridor 13). In this figure the section of the grooves is substantially identical. In this example, the cavity and groove assembly is surmounted by a closure plate (30) composed of a microscope slide 150 μm thick, thus forming a set of non-related cavities. The set of cavities and grooves has separation surfaces 20, 21, 22, 23 (extending by a surface 24) similar or different.
    A method, for example, for producing a microstructured substrate according to this figure consists in choosing for the material of thickness a microscope slide, for example of dimensions 26 mm × 76 mm made of plastic material such as polystyrene, to apply a pressure to the using a parallelepipedal metal part, on one of the surfaces of the lamella which will be the upper surface 17 of the micro-structured substrate, the lower surface 11 being supported by a flat metal part preferably covering the entire substrate. By heating the two metal parts to a temperature close to the glass transition temperature (95 ° C) of the material chosen and applying a pressure on the metal parallelepiped greater than 1 bar, the latter will be inserted into the polystyrene strip of some tens of microns depending on the time and the pressure exerted. After cooling the structure and separating the metal parts and the lamella, a rectangular-shaped surface cavity is obtained on the upper surface of the lamella constituting the analysis zone 12. This manufacturing method is generally likened to a method of thermoforming and it is compatible with most plastics and amorphous materials (such as glass, thermoplastic resins, etc.). In order to achieve the concentric grooves 13, 14, 15, 16 of the exhaust zone, it is possible, for example, to repeat the operation with four metal pieces of increasing rectangular sections positioned so as to form concentric grooves. For further details see J.L. Throne, Understanding Thermoforming, Hanser Gardner Publications, Inc., Cincinnati OH, 1999.
    The nested structure of the grooves makes it possible to control the zone of spreading of the liquid sample (1) between the microstructured substrate.
    Of course we can achieve the same structure according to the invention by depositing the contention barriers 20, 21, 22 and 23 on a support and thus create a relief.
    The convex surfaces formed by the edges delimiting the grooves 13, 14, 15, 16 form capillary barriers whose properties are inherent to the geometry and / or to the materials and surface treatments used to produce the support. According to the invention, the properties of these capillary barriers can be modified by modifying the geometry of the grooves, and in particular their edges and / or by treating them locally (partially or totally) and / or the surfaces which surround them, as this has has been explained above.
    FIG. 2 schematically illustrates the steps of filling the network of cavities / surface grooves during standard use by an experimenter, for example in biology. This example also illustrates another embodiment of a micro structured substrate according to the invention, in which the network of micro surface cavities is produced by a lithographic photo process as described, for example, in Sami Franssila's book "Introduction to Microfabrication ", Wiley in a layer of micro structured material covering a material of plane thickness. This manufacturing method has the advantage, compared to the thermoforming technique described above, of allowing to combine different materials in contact with the liquid sample (1).
    In FIG. 2a, the first step of filling the fine analysis cavity is represented: it corresponds to the deposition of the liquid sample (1) on the micro structured substrate. The user using, for example, a micropipette deposits a volume of liquid sample (1) greater than the sum of the volumes of the analysis zone 12 and the primary exhaust corridor 13. As a function of the affinity of the liquid for the material constituting the layer of micro-structured material, the lateral extension of the liquid sample is then either limited by the outer envelope of the analysis zone (negative capillary barrier or weak affinity), or by the inner envelope of the cavity forming the primary exhaust passage (13) or first groove (positive capillary barrier or strong affinity).
    To benefit from this contention effect, named before the capillary barrier, the volume of liquid delivered by the user must remain below a critical volume which depends on the affinity of the liquid sample with the layer of micro-structured material. In the case of an aqueous liquid sample (1), the use of a hydrophobic material such as an epoxy resin such as SU-8 resin (described as well as its implementation in US Patent 4882245 with the text is incorporated by reference in the present application) makes it possible to improve the retention force of the liquid sample during the liquid distribution step and therefore the maximum volume delivered. In this case of using a hydrophobic resin of this type which after removal of the insolated areas (the central cavity and the grooves) using a UV source of appropriate wavelength makes it possible to preserve the protected areas during the UV insolation step, thus creates negative capillary barriers between the outer contour of the cavity and the inner contour of the first groove, between the outer contour of the first groove and the inner contour of the second groove, etc. .., these negative capillary barriers all of the same kind in this example, being the separation surfaces 20, 21, 22, 23, 24, and the convex surfaces of the edges of the grooves attached thereto. The user then closes the assembly consisting of the micro-structured substrate, the liquid sample 1 and a lamella constituting the closure substrate 30 in order to finalize the forming of an analysis assembly comprising the substrate 10, the liquid 1 to be analyzed and the closure strip 30. The evolution of the liquid sample (1) within the holding structure is represented successively by FIGS. 2b, 2c and 2d on which the progressive lowering of the lamella 30 makes it possible to cause the liquid 1 to flow at least in the grooves 13 and 14 avoiding the formation of a bubble in the sample when the assembly is closed.
    For a critical approximation of the closure substrate 30 and the micro-structured substrate 10, the excess volume of liquid sample becomes too large to remain above the analysis zone 12. The pressure exerted on the liquid by the closure substrate 30 then pushes the liquid sample (1) to cross the capillary barrier bound to the surface 20 and fills the groove 13, which abruptly reduces the amount of excess liquid sample. The spreading zone of the sample then covers the analysis zone and the primary exhaust passage 13, the liquid being temporarily stopped by the new capillary barrier 21 between the primary 13 and secondary 14 corridors.
    When the closure substrate is again close (Figure 2c), the compression of the excess volume again reaches a tipping point which allows the filling of the second groove 14 (or first secondary exhaust passage). Subsequent filling of the nested secondary exhaust passages finally leads to the formation of a fine cavity as described in FIG. 2d, where in this example the last exhaust passage 15 (third groove) is only partially apparent. filled, depending on the excess volume remaining remaining just before the complete closure of the cavity 12. In this Figure 2, is taken as an example a substrate with three grooves 13,14 and 15, substantially identical sections.
    Examples of embodiments of the invention are described below. EXAMPLE 1
    In this example, the holding substrate 10 consists of a silica glass microscope slide on which has been deposited a microstructured layer of photosensitive resin SU8 of 50 μm in thickness defining an analysis zone (cavity). of total volume substantially equal to 5 μL on a circular surface of 1 cm 2 and surrounded by a series of two grooves, the first groove having a rectangular section of 50 μm in depth and 200 μm in width, a volume of 0.4 pL, (that is to say 0.08 times the volume of the cavity), the second groove having a depth of 50 μm and a width of 5 mm, ie a volume of 15 μL, the two grooves and the cavity being respectively separated from each other by a distance of 200 μm. It can be seen that, in general, the repetition of the grooves in accordance with the invention makes it possible to avoid the appearance of a bubble in the analysis zone as well for the small volumes of liquid (5 μL) as for the larger volumes (up to 20 pL in this example). The contact angle of the droplet delivered in the analysis zone is greater than 70 ° which characterizes a hydrophobic material (the SU-8 resin). This substrate example according to the invention allows the precise positioning and the maintenance of a cavity-like analysis zone, without bubble, preventing any overflow of the liquid sample outside the cavity of the micro-structured zone. This type of substrate makes it possible to control the thickness of the analysis cavity by controlling the height of the structured layer.
    In this example, while the hydrophobic material constituting the structured layer makes it possible to contain a larger excess volume than in the case of a hydrophilic structured layer, the use of a hydrophilic thickness material (a silica glass) allows on the other hand to improve the speed of filling successive grooves and thus minimizes the probability of bubbles forming in the grooves during a closing too fast on the part of the user. The filling of several grooves is particularly recommended to limit evaporation problems of the liquid sample in the analysis zone.
    Indeed, the succession of grooves filled by the excess liquid is a barrier to evaporation more effective than the number of cavities and cavity filling is large. It is another advantage of the invention to use a plurality of grooves which make it possible to obtain a liquid barrier surrounding the analysis zone and thus to prevent, as long as this liquid barrier is intact, evaporation in the zone. of analysis at the origin of fluidic or chemical instabilities. EXAMPLE 2
    The support of Example 1 was used to carry out an example of experiments showing that the spread zone of the liquid sample 1 on a substrate according to the invention is stable: the support assembly plus liquid sample trapped by the lamella 30 was subjected to heating at 50 ° C. for 30 minutes: no significant evaporation of the liquid sample in the analysis zone was observed: it was not affected by the evaporation process . In comparison, a slide - plate system without microstructures sees the same amount of liquid sample completely evaporated after only 20 min.
    The succession of grooves thus constitute a barrier to evaporation and keeps the analysis area intact.
    Without wishing to be bound by any theory, the inventors believe that this effect can be attributed to the succession of pockets of evaporation. Indeed, each groove, even empty, is a buffer zone where the humidity can increase until saturation. In the case of a simple barrier of contention, the evaporation is even faster in the analysis zone that the process is carried out by contact with the outside air directly. The slightest defect in the containment barrier or dust at this level also dramatically increases the evaporation of the liquid sample. In a system according to the invention, the multiplication of intermediate cavities between the analysis zone and the outside slows down the evaporation process. When it is desired to benefit from this advantage linked to the slowing down of the evaporation of the liquid, use will preferably be made of a support provided with a plurality of grooves with n greater than or equal to 3.
    Example 3
    This example describes the production of a microstructured substrate according to the invention comprising a plurality of surface cavities, each cavity being surrounded by a plurality of grooves (there is not necessarily although preferentially, the same number of grooves around each cavity). On a standard format strip (2.54 cm × 5.08 cm), for example made of PS or PMMA-type synthetic material, two parallel rows of four circular wells (cavities) 5 mm in diameter are produced by molding or stamping. 25 microns deep (ie a Vo volume of 0.5 microliter). Each well is distant from the previous well in the 5 mm row (distance between the perimeters of the cavities). Each well is surrounded by a series of 5 to 10 100 micron deep grooves 100 microns apart. A series of 5 grooves thus corresponds to a volume of 0.22 Vo and a series of 10 grooves corresponds to a volume of 0.48 Vo.
    This type of substrate makes it possible to deposit very small samples in a reliable manner, without the presence of bubbles after covering with the upper wafer, without any risk of contact between the samples and limiting the evaporation which can be rapid for these small volumes. This substrate allows in particular the simultaneous observation of several samples containing animal cells or yeast microorganisms, which have dimensions of between 2 and 20 microns.
    FIG. 3 illustrates an example of the filling process of the cavity using a liquid sample according to the prior art. A double restraining barrier 120, 121 is used for the liquid distribution of the sample 1 and for any sample leakage. The space 113 between the two containment barriers can be likened to a groove or primary exhaust corridor. The user deposits a volume of liquid greater than the volume limited by the containment barrier 120: the volume dispensed being greater than the volume of the analysis zone, during closure (FIG. 3b), the capillary barrier created by the barrier of compression contention 120 is exceeded by the liquid sample (1). By capillarity, under the influence of the angle between the upper substrate and the lower substrate, an uncontrolled amount of the liquid sample is absorbed in the space 113. The "wedge effect" between the closure substrate 30 and the micro-structured substrate causes the analysis zone to empty a large portion of the sample liquid. During the final closure of the fine cavity (FIG 3c), the analysis zone then has a bubble (5) due to the uncontrolled escape of the excess liquid. If the user dispenses a volume greater than the total volume of the analysis zone and the primary exhaust passage, the liquid is then caused to pass the restraining barrier 121: in addition to the leakage problems outside the device which represents a risk for the user and / or the instrument in the case of corrosive or carcinogenic samples, the uncontrolled escape of liquid out of the device is at the origin of bubbles in the device and therefore in the analysis zone. This situation is limited in the case of the invention (fig.l and fig.2) and in the case of the invention (fig.l and fig.2), however, it is important to note that it is Preferably, the primary corridor (the first groove) is completely filled before the escape step to the secondary corridors (second groove and subsequent grooves) to benefit from the best protection effect by the second groove. When positioning the closure strip, it is preferable to avoid a too fast movement or a sliding movement of the substrate 30 parallel to the micro-structured substrate, since this could cause the liquid to cross the surface 21 between the exhaust passage primary 13 and the secondary exhaust passage 14 before forming the negative capillary barrier on the surface 20 around the analysis zone 12. In this case, a bubble could be introduced uncontrollably into the zone of analysis.
    To eliminate this disadvantage, different variants are possible: The first solution is to reduce the volume of the first groove. The lower the volume, the shorter the time to fill it and the less the user's dexterity comes into play in the constitution of the capillary barrier. In particular, when the volume of the primary corridor 13 is at least 3 times, preferably at least 5 times and more preferably at least 10 times smaller than the volume Vo of the cavity 12, the filling time of the first groove 13 becomes negligible in front of the action of the user.
    The second solution consists in using a volume distribution for the different cavities and grooves which ensures that the analysis zone can not be emptied even in the absence of a complete negative barrier. Thus, if the volume of the primary exhaust corridor is smaller than the volume of the analysis zone and the volume of the adjacent secondary exhaust corridor, it is ensured that (i) the liquid prefers the cavities of low volume (strong affinity of the liquid for the surface of the substrate) and in this case the primary corridor will preferably be filled to the secondary corridor, (ii) either the liquid prefers the cavities of high volume (strong affinity of the liquid for the surface of the substrate ) and in this case the primary corridor will preferably be emptied with respect to the analysis zone.
    The third solution is to accelerate the filling of the first groove by playing on the materials inside the cavities and grooves. In the example of Figure 2, the glass used for the bottom of the cavity is hydrophilic which has the effect of attracting the liquid faster in the primary exhaust channel if the entire cavity surface had was made in a single hydrophobic material. On the other hand, the walls and the contact zones situated between the grooves must preferably remain hydrophobic, which makes it possible to improve the capillary barrier effect when filling the cavities. To improve the complete filling of a protective liquid barrier around the analysis zone, it is preferable that the hydrophilic surface portion in the primary exhaust channel completely surrounds the analysis zone. Conversely, for an oil-type liquid, it is more advantageous to provide a primary exhaust channel having a hydrophobic bottom and hydrophilic walls and contact zones. it is important to note that for an extension and a given total volume of the exhaust zone, the greater the number of exhaust corridors (preferably up to 5 grooves), the better the sample will remain correctly positioned things being equal.
    Figure 4 schematically shows a set of grooves in the form of micro channels of triangular sections (Figure 4a) or trapezoidal (Fig.4b). Indeed, the geometry of the various cavities and surface grooves constituting the micro structured network of the support can be advantageously modified for the needs of the user. The more the spacing between the exhaust passages is regular, the more the positioning will be reproducible. The use of a set of micro-channels of constant section for the exhaust passages also makes it possible to obtain a homogeneous filling of the exhaust zone and limits the filling errors related to a bad positioning of the closure substrate by the 'user. The use of surface cavities essentially consisting of portions of rectangular sections makes it possible to obtain angle effects at the bottom of the cavities that facilitate and accelerate the filling of the exhaust passages while limiting the appearance of bubbles. However, and depending on the affinity of the liquid sample for the substrate, it will be possible either to opt for sections of triangular type which reduce the probability of bubble formation (liquids of low affinity with the micro-structured substrate) or trapezoidal sections which increase the efficiency of the capillary barrier between the different cavities (liquids of high affinity with the micro-structured substrate).
    FIG. 4 illustrates two schematic examples of exhaust networks consisting of micro-channels of triangular and trapezoidal sections thus defined. It should be noted in these examples that the thickness of the surface cavities is not necessarily homogeneous. For example, the analysis zone may have a greater thickness than the exhaust passages when it is desired to save the liquid sample to be analyzed while benefiting from a maximum of exhaust passages for precise positioning of the sample. 'sample. Conversely, deepening the exhaust corridors will store more excess fluid to slow down the evaporation process.
    It is understood that several networks of surface cavities can be juxtaposed on the same substrate to dispense a larger number of samples.
    FIG. 5 represents an example of a substrate according to the invention intended for multi-sample analysis using surface cavities of circular lateral geometry. For each analysis zone 200, 201, 202, 203, 204, 205, an exhaust network consisting of a plurality of circular grooves arranged concentrically around each analysis zone, each network being independent and disjoined from the other networks allows to limit the contamination between the samples.
    Conversely, the analysis area can be organized as the related juxtaposition of several areas.
    Finally, it should be noted that the closure substrate (lamella 30) or the micro-structured substrate, can be constituted by any system having a flat surface, respectively a top surface, including analysis (CCD sensor, etc.). or control (microfluidic thermalization or infusion chip).
    The system described by the invention can be used to position a biological sample in the form of a fine cavity before observation by optical microscopy. Another example of application consists in using the system of the invention for a biological detection system by interaction with objects distributed on the surface of the substrate (DNA or protein chip) in which the invention makes it possible to close the chamber reaction by ensuring a homogeneous reaction throughout the analysis zone. A third example of application is the systematic analysis of a chemical using an automatic dispensing system coupled with a transmission analysis using a fine cavity spectroscopy system formed at the same time. aid of the invention without leakage of the sample.
    权利要求:
    Claims (14)
    [1" id="c-fr-0001]
    1 - Substrate (10) micro structured liquid sample support comprising a lower face (11) and an upper face (17) which comprises on the one hand, at least one surface cavity (12) of volume VO opening on said upper face (17) and forming an analysis zone of the liquid sample and secondly, a first groove (13) of a first volume VI, arranged around each surface cavity (12) and opening on its upper face (17), characterized in that it further comprises at least one second groove (14,15,16) opening on its upper face and arranged around the first groove (13), the sum of the volumes of the second grooves being equal to V2, the volume V1 + V2 being greater than or equal to 0.05 VO, preferably 0.1 VO.
    [2" id="c-fr-0002]
    2 - Substrate according to claim 1, characterized in that the volume V1 + V2 is greater than or equal to 0.2 VO, more preferably greater than or equal to 0.3 V0.
    [3" id="c-fr-0003]
    3 - Substrate according to one of claims 1 or 2, characterized in that it comprises in total n grooves (13,14,15,16), with n> 2, each groove of rank p greater than or equal to 2 and less than or equal to n being disposed around the row groove (p-1), the total volume V3 of the (n-1) first grooves (13,14,15) being greater than or equal to 0,05 VO.
    [4" id="c-fr-0004]
    4 - Substrate according to one of the preceding claims, characterized in that it comprises n grooves (13,14,15,16), n being less than or equal to 100, preferably less than or equal to 10.
    [5" id="c-fr-0005]
    5 - Substrate according to one of the preceding claims, characterized in that the section of each of the grooves (13,14,15,16) is substantially identical, preferably constant.
    [6" id="c-fr-0006]
    6 - Substrate according to one of the preceding claims, characterized in that it comprises n concentric circular grooves (13,14,15,16) of the same section, preferably arranged at equal distance from each other.
    [7" id="c-fr-0007]
    7 - Substrate according to one of the preceding claims, characterized in that the section of at least one of the grooves (13, 14, 15, 16), preferably the nth or last groove (16), the outermost, is greater than that of the other grooves, preferably greater than or equal to 5 times, more preferably 10 times the section of the (n-1) th groove.
    [8" id="c-fr-0008]
    8 - Substrate according to one of the preceding claims, characterized in that the depth of the grooves is between about 1 nanometer and 500 microns, preferably between 20 microns and 500 microns.
    [9" id="c-fr-0009]
    9 - Substrate according to one of the preceding claims, characterized in that the distance between the inner contour of a groove (14,15,16) and the outer contour of the previous groove (13, 14, 15) that it surrounds and / or the cavity (12) is between about 1 micron and 1 mm, preferably about 100 microns.
    [10" id="c-fr-0010]
    10 - Substrate according to one of the preceding claims, characterized in that at least some areas of the upper face (17) and / or the cavity (12) and / or grooves are treated so as to modify in these areas the properties of the capillary barrier inherent in the surface state of the material constituting the substrate and / or the geometry of the grooves and surfaces.
    [11" id="c-fr-0011]
    11 - Substrate according to one of the preceding claims, characterized in that it has at least six, preferably at least eight surface cavities, each cavity having a diameter of about 5 mm and a depth of between 20 micron and 500 microns and being spaced approximately 5 mm from another cavity, each cavity being surrounded by a series of 5 to 10 grooves of about 100 microns in depth, spaced from each other by about 100 microns.
    [12" id="c-fr-0012]
    12 - Substrate according to one of the preceding claims, characterized in that the substrate (10) is constituted by a monolayer or multilayer material in which are etched or thermally, mechanically and / or chemically formed at least one of the grooves (13). , 14, 15, 16) and / or the cavity (12).
    [13" id="c-fr-0013]
    13 - Analysis set of a liquid sample, characterized in that it comprises a substrate (10) according to one of the preceding claims, on which at least one liquid sample (1) is deposited in at least one cavity (12). ) of the substrate (10) which is covered by a lamella (30) closing all the cavities (12) of the substrate (10).
    [14" id="c-fr-0014]
    14 - Use of a substrate according to one of claims 1 to 12 for carrying out analysis of a liquid sample.
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    同族专利:
    公开号 | 公开日
    EP3347128B1|2021-01-27|
    US20180250670A1|2018-09-06|
    FR3040895B1|2020-01-10|
    WO2017042115A1|2017-03-16|
    US10471430B2|2019-11-12|
    EP3347128A1|2018-07-18|
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    法律状态:
    2016-08-22| PLFP| Fee payment|Year of fee payment: 2 |
    2017-03-17| PLSC| Search report ready|Effective date: 20170317 |
    2017-10-05| PLFP| Fee payment|Year of fee payment: 3 |
    2019-09-30| PLFP| Fee payment|Year of fee payment: 5 |
    2020-09-30| PLFP| Fee payment|Year of fee payment: 6 |
    2021-09-30| PLFP| Fee payment|Year of fee payment: 7 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1501889|2015-09-11|
    FR1501889A|FR3040895B1|2015-09-11|2015-09-11|LIQUID SAMPLE SUPPORT SUBSTRATE, ASSEMBLY COMPRISING SUCH SUBSTRATE AND ITS USE|FR1501889A| FR3040895B1|2015-09-11|2015-09-11|LIQUID SAMPLE SUPPORT SUBSTRATE, ASSEMBLY COMPRISING SUCH SUBSTRATE AND ITS USE|
    US15/759,218| US10471430B2|2015-09-11|2016-09-05|Substrate for supporting liquid sample, an assembly comprising such a substrate and use thereof|
    EP16760485.9A| EP3347128B1|2015-09-11|2016-09-05|Assembly comprising liquid sample support substrate and use thereof|
    PCT/EP2016/070805| WO2017042115A1|2015-09-11|2016-09-05|Liquid sample support substrate, assembly comprising such a substrate and use thereof|
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