Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
  • B01L3/50255—Multi-well filtration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
  • B03C5/02—Separators
  • B03C5/022—Non-uniform field separators
  • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0819—Microarrays; Biochips
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications

Definitions

• ELECTRODOT METHOD FOR ANALYZING BIOLOGICAL SAMPLES IN SPOT MATRIX FIXED BY THE ACTION OF AN ELECTRICAL CURRENT
• Matrix or dot plot methods are conventional methods of analyzing a plurality of biological samples, such as:
• the implementation of the dot plot filtration generally involves devices comprising a pierced plate transverse wells, such as a biological sample in solution or suspension is disposed in each well of the plate, said plate being arranged contiguously on a filtration membrane.
• a pressure difference ⁇ applied on both sides of the membrane, allows the filtration of the biological sample through said membrane so as to retain on the latter targets of interest samples at the same time as the other elements of the samples, each sample forming a spot of a spot matrix of mirror geometry to that of the well plate.
• the targets of interest of each spot will then be analyzed, by different methods of analysis used in biology, such as: Fluorescence imaging, chemiluminescence, colorimetry methods, when the targets of interest are labeled before, during or after filtration by a fluorescent, chemiluminescent or colorimetric marker, such as for example the marker is integrated into the structure of target or interact with the target structure, including the use of antibodies directly labeled or labeled with a secondary antibody;
• Spectroscopy methods such as LIBS (Laser Induced Plasma Spectroscopy), mass, infrared, etc. ; or any other method that quantifies or identifies the interest target (s).
• dot blot technologies by filtration is largely limited by the methods of sample preparation. Indeed, the addition of chaos-tropes agents such as urea or detergents such as sodium dodecyl sulfate (SDS), tritone, etc., in the sample to extract interest targets, strongly penalizes the capture by the filtration membranes of many nucleic acid or protein targets.
• chaos-tropes agents such as urea or detergents such as sodium dodecyl sulfate (SDS), tritone, etc.
• the wells of the filtration plates must have a certain minimum volume allowing the detection of the targets as a function of the detection sensitivity, which results in a dispersion and / or a separation of the spots on the screen.
• filtration membrane directly proportional to the well size of the well plate.
• the surface area of the membranes is large relative to the number of spots, and then requires for the analysis of large volumes of saturation and revealing solutions comprising expensive compounds such as labeled antibodies, unlabeled antibodies and labeled secondary antibodies. etc. ;
• the filtration is not well-independent, therefore when the samples have different viscosities from one well to another, the filtration rate varies from well to well.
• the conditions and the filtration rate are no longer controlled as soon as a filtration well is emptied, since ⁇ applied to the membrane drops as soon as the first well of the plate is empty.
• the cross-affinity problems that may arise for antibody markings, for example directed against certain forms of protein phosphorylation.
• Fixation membrane means any membrane or substrate such as:
• the present invention relates to a method for fixing, on a spot matrix, all or part of a plurality of biological samples such that each sample is placed in a well Figl .l of a transverse well plate. and opening F 1.2, and such that is positioned under said well plate fixing membrane F 1.3, so as to subject each sample to the simultaneous or sequential action, an electric field Figl .4 and a pressure Figl .5 such that the electric field has a direction perpendicular to the fixing membrane, and a potential differential between the two faces of the fixing membrane of between 1 milivolt and 1 kilovolt (preferably between 10mV and 100V, and more preferably between lOOmV and 10V), and such that the pressure has a differential pressure ⁇ between the two faces of the fixing membrane of between 0.1 millibar and 100 bar (preferably between lmb and 10b, and more preferably between 10mb and 10b).
• the fixation of the molecules of interest on the attachment membrane is by the double action of the electric field and the filtration.
• the action of the electric field allowing a better fixation of the targets of interest on the membrane even in the presence of a detergent in the medium such as the SDS, the triton or other chaostropic agents, or any combination thereof. agents and detergents.
• the electric field is achieved by two electrodes of conductive materials, such as conductive polymer, conductive composite, metal, gold, platinum, tantalum, etc., disposed above Figl .6 and below Figl. 7 of the well plate, the fixing membrane being interposed between the lower face of the well plate Fig2.14 and the lower electrode Figl .7.
• conductive materials such as conductive polymer, conductive composite, metal, gold, platinum, tantalum, etc.
• At least one of the electrodes will be a sheet of an electrical conductor such as gold, platinum, copper, tantalum, etc., optionally deposited on a polymer such as kapton, silicone, PMMA, PLA. ...
• the metal electrode sheet will comprise at least one tacky structured surface, obtained for example by means of a repositioning adhesive, or at least one joined structural surface FIG. 9, for example made of rubber, silicone, or any other polymer, such as the tacky surface, leaves the surfaces of the Fifl-shaped spot electrode 10 free from the well plate wells so as to have an electrode array exposed metal.
• the spots of the electrode left free by the contiguous or sticky surface will comprise at least for the lower electrode pores for letting a liquid pass.
• At least one of the faces of the fixing diaphragm FIG. 3 will comprise a joined or sticky structured surface 9 leaving a matrix of fixing membrane spots Fig. 11 opposite the well matrix.
• a porous paper, forming a Fig. 8 pad for example 0.1 to 5 mm thick such as wattman paper, laid paper etc., is disposed against at least one of the electrodes, in order to make a buffer between the electrode and the membrane, or the electrode and the well plate.
• at least one side of the paper will be structured by a glue or a joined polymer to leave free a paper spot matrix Fig. 12 vis-à-vis the well matrix of the well plate.
• the well plate is made such that the bottom face Fig2.14 of the well plate as well as the lower diameter of the wells (face of the well plate receiving the binding membrane) are reduced for example by a homothety of a factor K and / or k 'relative to the size of the upper face Figl .13 of the well plate and the upper diameter of the wells.
• This reduction of a factor K and / or k ' makes it possible to reduce the spot size by a combination factor K and k' but also, the distance between the spots of the same combination factor K + k 'and makes it possible to achieve reduced binding membranes of K-factor relative to the upper surface of the well plate.
• the well plate has a liquid ion electrode, gel or impregnated sponge, connecting the upper face to the lower face of the well plate. This electrode makes it possible to impose a potential difference between the two faces of the membrane whatever the level of sample in the wells of the matrix.
• the upper electrode will comprise Fig. 6, the upper face of the well plate, and a portion of the well surface.
• a Fig2.15 structure forming an electrode such as a cross or any other shape connected to the upper electrode will be disposed in a transverse plane of the wells of the well plate.
• the pressure difference ⁇ is obtained by suction below the attachment membrane through the lower electrode.
• the stack of all or part of the elements described above for example is arranged on a suction grid Fig3.16 below which is applied a vacuum.
• the grid Fig3.16 is structured by an adhesive or a joined material, so as to delimit tight spots between them where said grid appears free to form a matrix of grid spots vis-à- screw wells of the well plate.
• the suction grid Fig3.16 and the lower electrode are combined, the grid then comprises at least one electrically conductive element, for applying an electric field, in each well of the well plate, through the fixing membrane.
• the electrically conductive element may be a plating or a metallic deposit such as gold, copper, tantalum, platinum, etc.
• the pressure difference ⁇ is obtained by a pressure applied above the membrane through the wells of the well plate and the upper electrode.
• the biological samples are introduced into the wells 1 of the well plate by means of a plate plate Fig3.18 forming a matrix of wells arranged in a manner homologous to the well of the well matrix.
• the samples are introduced into the plate of boxes kept horizontal, such as for example a sample per box.
• the well plate comprising any combination of the stacks described is clipped Fig3.19 on the plate plate remained horizontal, the upper face of the well plate being close to the openings of the plate of boxes.
• both well plates and well plates may be guided and held against each other by magnet and magnetic or ferrous platinum systems, introduced for example at the four corners of the plates.
• the box plate comprises in each box a movable piston Fig3.20, for example flexible polymer such as silicone, rubber etc. Behind each piston is disposed in the box plate, a pore Fig3.21 allowing, by displacement of the piston, to compensate the pressure or to apply pressure, depending on whether the device is used in overpressure or depression.
• the movable piston guarantees a homogeneous application of the pressure or of the depression, during the process as soon as a well is emptied it is obstructed by its piston which prevents the fall of ⁇ .
• the various electrodes and membranes used in the device will comprise tabs 22 allowing rapid recovery of the fixing membrane and the different surfaces of the well plates, plate of boxes, electrode, paper pad etc. are structured 9 by a sticky material, joined to define a matrix of spots in the image of the well matrix such that each spot is isolated from other spots by said material.
• the device will comprise a rapid revelation kit.
• the kit will comprise for each fixing membrane, a plastication complexing bag of sufficient size to receive the membrane, the bag will be provided with a rapid closure device such as closing slide or closing slider.
• One of the faces of the bag will comprise a fast attachment device to a support, for example the velvet part of a fastening or self-gripping strip, the hook portion of said strip being arranged on the support receiving the bag for example in a incubation oven.
• the bag will preferably be opaque protecting light, for example black and opaque to ultraviolet, so that the light can not alter the properties of the reagents and biological material disposed in said bag.
• the bag will be pre-filled with a solution or with a powder that makes it possible to produce a solution for saturating the membrane.
• the bag will contain from 0.1 to 10g of lyophilized milk powder.
• the kit will include, for each attachment membrane, a rapidly opening hermetically sealed capsule made of aluminum, plastics material, etc., containing freeze-dried labeling reagents such as lyophilized antibodies and saturation elements such as lyophilized milk. The contents of the capsule will preferably be compressed to obtain an aggregated body.
• the kit will also comprise a stirring furnace thermostatted between 4 ° C and 50 ° C, preferably 37 ° C, the oven will include elements to fix the revealing bag and allow it to stir.
• the kit may be formed by a combination of all or part of the aforementioned elements.
• the stirring system of the furnace may for example comprise a rotating axis with a diameter of between 0.5 cm and 60 cm, said axis having means for fixing the revelation bag, for example the hook part of a Velcro tape.
• the fast opening hermetically closed capsule will have at least one antibody directed against a target of interest, said antibody being labeled with at least one fluorophore, chromophore, bioluminescent agent or chemiluminescent, such that each antibody directed against a given target is labeled with a fluorophore, a chromophore, a given bioluminescent or chemiluminescent agent.
• the rapid-opening hermetically sealed capsule will have at least one primary antibody directed against a target of interest and a secondary antibody directed against the at least one primary antibody such that the secondary antibody is labeled with less a fluorophore, a chromophore, a bioluminescent or chemiluminescent agent.
• the fast opening hermetically closed capsule will comprise at least one unlabeled saturation antibody, neither directly nor by a secondary antibody, making it possible to compete with parasitic aspecific bonds of a given primary antibody to other targets than its target of interest.
• the capsule will comprise primary antibodies labeled, directly or by a secondary antibody, directed against phosphorylated proteins of interest such as ⁇ 2 ⁇ histone, Phospho KU 70, phospho ATM, etc., and at least one of the unmarked saturation antibodies. directed against phospho-serine, phospho-trreonine, phosphothyrosine, etc., such that the aspecific attachment of primary antibodies to phosphorylated targets different from their target of interest is competed by the binding of the saturation antibodies.
• the implementation of the kit will include six main steps:
• Solubilization of the saturation agent contained in the complexation bag by adding a dose of buffer, for example 50 ml of TBS per 2.5 g of milk.
• Fig4.23 Example of extraction of the biological material, for HEK cells, by various extraction buffers 25, 26, 27, 28, in dot-blot filtration then labeling with a primary anti-histone antibody ⁇ 2 ⁇ and a secondary chemo antibody -Luminescent directed against the primary antibody.
• Fig4.24 Example of extraction of the biological material, for hair follicles, by various extraction buffers 25, 26, 27, 28, dot-blot filtration and labeling with a primary anti-histone antibody ⁇ 2 ⁇ and a chemiluminescent secondary antibody directed against the primary antibody.
• Fig4.29 Example of extraction of the biological material, for HEK cells, with a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary anti-histone ⁇ 2 ⁇ antibody and a secondary chemiluminescent antibody directed against primary antibody, the labeling products being lyophilized in an aluminum capsule.
• Fig4.30 Example of extraction of the biological material, for hair follicles, by a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary anti-histone ⁇ 2 ⁇ antibody and a secondary chemiluminescent antibody directed against primary antibody, the labeling products being lyophilized in an aluminum capsule of the freeze-dried skimmed milk.
• Fig4.31 Example of extraction of the biological material for HEK cells, by a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary antibody anti-histone ⁇ 2 ⁇ antibody labeled with a fluorophore at 667 nm and a primary antibody anti-histone H2AX labeled with a fluorophore at 488 nm, the labeling products being lyophilized in an aluminum capsule containing freeze-dried skimmed milk.
• the invention relates to a method for fixing, on a spot matrix, all or part of a plurality of biological samples such that each sample is placed in a well (1).
• the invention also relates to a device for implementing the above method comprising the transverse and through well plate (2) and the fixing membrane, characterized in that it comprises means for submitting each sample to the simultaneous, sequential action of an electric field (4) and a pressure, these means comprising two electrodes supplying the electric field, said electrodes being composed of conductive materials, such as for example a conductive polymer, a conductive compound, gold, platinum, or tantalum, disposed above (6) and below (7) of the well plate (2), the attachment membrane being interposed between the underside of the well plate and the lower electrode.
• conductive materials such as for example a conductive polymer, a conductive compound, gold, platinum, or tantalum
• the device may include one or more of the following features, taken separately from each other or in combination with each other:
• At least one of the electrodes is a sheet of an electrical conductor, which comprises at least one adhering structured surface (9), the structured surface leaving free the surfaces of the spot-shaped electrode (10) in to the wells of the well plate, so as to have a matrix of apparent metal electrodes, the spots (10) of the electrode left free by the surface contiguous or adhesive comprising at least for the lower electrode pores for passing a liquid;
• a porous paper forming a pad (8) 0.1 to 3 mm thick such as for example a wattman paper, or a laid drawing paper, is placed against at least one of the electrodes and that at least one face paper is structured (9) by an adhesive or a joined polymer so as to leave a paper spot matrix (12) free from the well matrix of the well plate;
• the lower face (14) of the well plate as well as the lower diameter of the wells, defined as the face of the well plate receiving the fixing membrane are reduced in size by a homothety, such as a factorial homothetry;
• K a combination of factor K and k 'with respect to the size of the upper face (13) of the well plate and the upper diameter of the wells, said reduction making it possible to reduce the size of the spots by distance between spots on the attachment membrane;
• the well plate has a liquid ion electrode, gel or impregnated sponge, connecting the upper face to the lower face of the well plate;
• the upper electrode comprises, the upper face of the well plate, part of the surface of the wells, and optionally a structure (15) disposed in a transverse plane of the wells of the well plate; - a suction grid (16) and the lower electrode (7) coincide, such that said grid comprises at least one electrically conductive element for applying an electric field, in each well of the well plate, to through the fastening membrane; the pressure difference ⁇ is obtained by suction (17) below the fixing membrane through the lower electrode of the suction grille, below which a vacuum is applied; the pressure difference ⁇ is obtained by a pressure applied above the membrane through the wells of the well plate and the upper electrode; a box plate (18) forming a matrix of wells arranged in a manner homologous to the wells of the well matrix, is used to introduce the biological samples into the wells of the well plate, and comprises in each box a movable piston ( 20) preferably of flexible polymer, such as for example silicone rubber, so that behind each piston is disposed in the plate of boxes, a
• the electrodes, membranes, buffer papers used of the device will comprise tabs (22) allowing rapid recovery of the fixing membrane and that the different surfaces of the well plates, box plates, electrodes, buffer papers are structured by a sticky, joined material defining a matrix of spots in the image of the matrix of wells such that each spot is isolated from other spots by said material;
• the present invention further relates to a rapid revelation kit comprising the device.
• the rapid revealing kit comprises, for each fixing membrane, a plastication complexing bag of sufficient size to receive the membrane, said bag is provided with a rapid closure device such as for example a slide, or a slider, one of the faces of the bag comprises a device for fast attachment to a support, such as for example the velvet part of a self-gripping strip, said bag is preferably opaque protecting from light and is pre-filled with a powder allowing to achieve a solution of saturation of the fixing membrane such as 0.1 to 10g of freeze-dried milk powder, the kit also comprises for each attachment membrane a hermetically closed fast opening capsule, made of aluminum, plastic, which contains reagents freeze-dried labeling and saturation such as antibodies, saturation elements of the binding membrane, fluorophore, chromophore, a chemiluminescent bioluminescent agent, the contents of the capsule being preferentially compressed so as to obtain an aggregated body;
• a hermetically closed fast opening capsule made of aluminum, plastic, which contains reagents freeze-dried label
• a stirring system of a complexing furnace comprises a rotating axis with a diameter of between 0.5 cm and 60 cm, said axis having means for fixing the revealing bag, such as for example hooks of a band self-gripping;
• the hermetically closed rapid-opening capsule comprises at least one primary antibody directed against a target of interest and at least one secondary antibody directed against the at least one primary antibody such that said secondary antibody is labeled with at least one fluorophore, a chromophore, a bioluminescent or chemiluminescent agent;
• the hermetically closed fast-opening capsule comprises at least one saturation antibody, making it possible to compete with the nonspecific parasite bonds of a primary antibody for targets other than its target of interest.

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• 2015
  • 2015-03-03 FR FR1500399A patent/FR3033409B1/fr not_active Expired - Fee Related
• 2016
  • 2016-03-03 EP EP16713963.3A patent/EP3265230B1/de active Active
  • 2016-03-03 WO PCT/FR2016/050477 patent/WO2016139424A1/fr active Application Filing

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