EP3436194A1 - Membranes d'analyse de dispositifs microfluidiques, réalisées en un matériau en fibre de verre - Google Patents
Membranes d'analyse de dispositifs microfluidiques, réalisées en un matériau en fibre de verreInfo
- Publication number
- EP3436194A1 EP3436194A1 EP17713317.0A EP17713317A EP3436194A1 EP 3436194 A1 EP3436194 A1 EP 3436194A1 EP 17713317 A EP17713317 A EP 17713317A EP 3436194 A1 EP3436194 A1 EP 3436194A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analysis
- channels
- analysis membrane
- zone
- fiberglass
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- 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
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- 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
- B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
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- 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
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/16—Reagents, handling or storing thereof
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- 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
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/069—Absorbents; Gels to retain a fluid
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- 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
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/087—Multiple sequential chambers
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- 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
- B01L2300/12—Specific details about materials
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- 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
- B01L2300/12—Specific details about materials
- B01L2300/126—Paper
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- 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
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
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- 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
- B01L2400/08—Regulating or influencing the flow resistance
- B01L2400/084—Passive control of flow resistance
- B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
Definitions
- the present invention relates to the general field of microfluidics. It relates more particularly to microfluidic devices for diagnostic purposes, as well as the manufacturing processes of such devices.
- Microfluidics can be defined as the study of phenomena that govern the movement of small volumes of fluid, especially liquid. It encompasses the development of systems and devices for circulating and / or manipulating small volumes of fluids for a variety of purposes, for example:
- Micro fluidics thus finds applications in many technical fields.
- diagnostic tests using microfluidic devices have undergone a rapid evolution since the last two-three decades (Yetisen et al., 2013 - Lab Chip, 2013 (13) 2210-2251: "Paper -based microfluidic point-of-care diagnostic devices ").
- microfluidic sensors these devices not only make it possible to analyze small volumes of liquid samples, they also make it possible to undertake, on a single analysis platform, a plurality of detection tests, or even of quantification, analytes and / or target pathogens by simple and rapid manipulation.
- the present invention is more specifically concerned with disposable microfluidic devices, of the type of those which comprise an analysis membrane made of a porous material and which operate according to a so-called fluid lateral movement principle, in this case liquids .
- the general principle of operation of this type of microfluidic device is essentially based on an analysis membrane made up / fabricated from a sheet of porous material, hydrophilic and absorbent, on which and in the bulk of which is formed a hydrophilic network.
- a liquid to be analyzed for example a liquid biological sample
- a liquid biological sample once deposited on said analysis membrane, can progress by simple capillarity and be submitted an analysis or a series of qualitative and / or quantitative analyzes of its constituents (immunodetection, molecular detection, affinity test, ligand-receptor coupling test, pH evaluation ).
- deposit zones at which the liquid to be analyzed is deposited
- reaction zones at which the detection and / or dosing reactions take place
- reservoir zones at which reagents and / or additives necessary for the reactions to be carried out are stored, generally in dry form before being rehydrated (or solubilized) and transferred to specific reaction zones,
- analysis membranes for microfluidic devices made from a sheet of porous material, hydrophilic and absorbent, are shaped mainly according to three modes of design.
- these analysis membranes have a two-dimensional shape which can be described as solid form and their general geometry is relatively simple (for example, a rectangle, a square, a disc ).
- hydrophilic networks deposition zones, reaction zones, mixed zones and / or reservoir zones, fluidic conduction channels
- hydrophilic networks are traced and circumscribed by means of structures made of solid (s) and hydrophobic material (s) ), implanted through the very thickness of the analysis membrane and thus forming liquid-tight barriers.
- the analysis membranes have a two-dimensional shape that can be described as pruned or cut form.
- Such analysis membranes are cut from a sheet of porous, hydrophilic and absorbent material, precisely following the outer contour of the hydrophilic network formed by the different areas of interest and the fluidic conduction channels.
- the analysis membranes In a mixed mode, notably described by Fan et al., 2013 (Nano / Micro Engineered and Molecular Systems (NEMS), 2013 - 8 th IEEE International Conference: "Low-cost rapid prototyping of flexible plastic paper based microfluidic devices”), the analysis membranes have a shape that can be described as solid form. These analysis membranes are shaped / machined from a sheet of porous, hydrophilic and absorbent material, the underside of which is coated with a mechanical reinforcing film. On these analysis membranes, the hydrophilic network is drawn and delimited not by impervious barriers implanted through the thickness of the analysis membrane itself, but by the vacuum left after a porous and hydrophilic material removal. This removal of material can be achieved using a variety of machining techniques, including etching, ablation.
- the laser allows a fast and precise implementation of these machining techniques.
- One of these approaches is to optimize the typography of the analysis membranes, in particular to reduce the distances of delivery of the liquid to be analyzed to the reaction zones.
- Another approach aims to improve the performance of existing cellulose and nitrocellulose substrates, for example by functionalizing the fibers, and / or to propose new fibrous materials with improved properties in terms of hydrophilicity and / or absorption and / or of conduction of fluids.
- cellulose fiber-based materials including cotton and linter
- glass silk, viscose, polypropylene, polyester, polyamide (Nylon ® ), poly (lactic acid) or PLA.
- Said fibers may optionally be functionalized and / or loaded and / or doped with additives (for example, talc, diatomite, etc.).
- Fang et al, 2014 (Lab Chip, 2014 (14) 911-915: "Paper-based microfluidics with high resolution has a glass fiber membrane for bioassays”) describes an analysis membrane, of pruned shape, entirely made of absorbent material and liquid diffuser, of fiberglass composition. This is shaped according to the desired configuration, by a mechanized cutting of a sheet of fiberglass using a Cricut Expression ® cutter (PROVO CRAFT @ NOVELTY, USA), said fiberglass sheet having been previously laminated on a PVC reinforcement film.
- a fiberglass material is preferred here to the (nitro) cellulose fiber materials, for its greater hydrophilicity and greater wettability, which give rise to a much better conductance and conductivity to liquids.
- the analysis membrane is configured with a central circular zone forming deposit zone for receiving a liquid sample to be analyzed. From this deposition zone radiate eight channels, each leading to a peripheral circular zone of smaller circumference and forming a reaction zone.
- these reaction zones are prepared for the evaluation of pH, the detection of glucose, various proteins, nitrites, ketone bodies, etc.
- the reagents necessary for different detections are deposited in the form of solutions in the corresponding reaction zones. The drying of the membrane takes one hour, under ambient conditions.
- each reaction zone is independently shaped in the material chosen (for example by wax impregnation, by etching or by cutting) and then functionalized separately from the other components of the membrane analysis achieve. Only after fixation / adsorption of the reagents, the reaction zones are assembled to the rest of the analysis membrane.
- the analysis membrane is formed by assembling two pieces of different materials; one is cut from a fiberglass sheet (ie, one MF1 filter paper) and the other from a cellulose fiber sheet (in this case a Whatman TM No. 1 filter paper).
- a deposit area In the fiberglass part, there is a deposit area. At the junction between the two fibrous parts, this deposit zone opens on a channel formed in the cellulose fiber portion. This main channel progresses through the cellulose fiber portion before dividing into two secondary channels, each leading to a reaction zone.
- the zones of interest and the channels of the analysis membrane are traced and delimited with solid wax by a method of impregnation with wax.
- the fiberglass material was chosen for its ability to filter whole blood and allow separation between plasma and blood cells. The cells are retained in the fiberglass deposition zone, while the plasma diffuses out of the fiberglass towards the cellulose fiber portion and then through the traced channels before reaching the reaction zone.
- the cellulose fiber material was chosen for its fluidic conduction properties allowing a good transfer of the plasma from the deposition zone to the reaction zones.
- the microfluidic device described by EP 2,226,635 has been specifically designed for the immunodetection of Mycobacterium tuberculosis.
- the analysis membrane of this device is in the form of a linear succession of parts made from different hydrophilic and porous materials, cut and assembled to each other to form a strip. According to a particular embodiment, from upstream to downstream of the analysis membrane and adhering to the adhesive side of an adhesive strip, are arranged successively and partially overlapping:
- the porous material constituting this element may be a woven or a nonwoven, polyethylene, polypropylene, cellulose, preferably a filter paper;
- transient storage zone element for a labeled antibody, directed against the MPB64 protein (an analyte indicating the presence of Mycobacterium tuberculosis); this element is made of a nonwoven material of fiberglass composition; a little further downstream is a capture zone at which an antibody directed against this same MPB64 protein is immobilized in the same thickness of the nitrocellulose membrane;
- an absorbent element made of a material capable of rapidly absorbing a liquid.
- the present invention aims to overcome the disadvantages encountered in the design and manufacture of microfluidic device analysis membranes known to date. More particularly, it aims to provide new analysis membranes, structure and design, compatible with the constraints of an industrial operation of a single-use diagnostic device, particularly in terms of cost of production and profitability.
- the present invention provides a microfluidic device analysis membrane, formed integrally from a sheet of absorbent material and liquid diffuser, of fiberglass composition.
- said analysis membrane comprises:
- At least one zone called deposit zone
- reaction zone in which at least one reagent is adsorbed directly to said fiberglass liquid-diffusing material, or indirectly via a coupling agent
- this analysis membrane comprises at least one reaction zone circumscribed in a space of the analysis membrane, inside which, the channels that arrive upstream and / or the channels that leave again downstream:
- a mechanical reinforcing layer made of a hydrophobic and impermeable material, for example a plastic film of poly (ethylene terephthalate) type, or PET.
- analysis membrane refers to the main element of a microfluidic device for diagnostic purposes which, by its different constituent parts, ensures the reception of the liquid sample to be analyzed and the delivery of least part of the constituents of this sample to areas of analysis / detection, and serves as a support structure for the implementation of these analyzes / detections.
- said analysis membrane is defined as being made in one piece, that is to say that it is formed of a single piece, continuous and without interruption, made in a unique material. That being so, it may be envisaged to add to an analysis membrane in one piece according to the invention of the reported elements, secondary functionality (s).
- absorbent material and liquid diffuser refers to a porous textile whose structure consists of an ordered or random assembly of fibers (that is, a woven or a nonwoven). This textile has the ability not only to absorb aqueous liquids but also to diffuse them through its structure.
- the absorbent material and liquid diffuser used in the making of the analysis membranes is of glass fiber-based composition.
- fiberglass material can be advantageously used in the present description.
- Such fiberglass materials are usually encountered in analytical laboratories (both medical and industrial) where they are commonly referred to as “fiberglass filter media” or “fiberglass filters”, and are typically applied to the treatment of liquid compositions for separation and purification purposes.
- Their manufacture consists of forming sheets / membranes with glass micro-fibers arranged in a more or less random or orderly manner, then of joining them chemically and / or thermally and / or mechanically.
- - WHATMAN TM filters GE Healthcare Life Sciences, USA
- MF1, LF1, VF1, VF2 and Fusion 5 for whole blood filtration and cell / plasma separation
- the GF filter / C for the separation of solid substances suspended in a fluid
- reagent is used in this broadly defined description to refer to any substance used to implement the intended assay (s) / detection (s), which this substance interacts directly with or not with the target (s) sought or intervenes only as an auxiliary.
- the reagents applied to the present invention may be, for example:
- antibodies capture and / or detection
- haptens aptamers
- markers markers, coupling agents, spacer arms, adapters ...
- nucleic probes and / or nucleic primers labeled or not, enzymes (such as enzymes with activity polymerase, recombinase or helicase), labeled or unbranched nucleic acids, optionally spacer arms, adapters ...
- space where the rate of diffusion of liquids is slowed down refers to a particular portion / part / region of the test membrane at which the constituent fiberglass material of said membrane has been locally modified in its composition and / or in its structure and / or configuration to slow the diffusion rate of the liquids and / or to lengthen their path between two points, in this case between two reaction zones.
- the time required for a liquid to travel the distance between two points in such a space is significantly greater than it would take to travel the same distance in another portion of the analysis membrane.
- the expression "space with slow diffusion" can be advantageously used in the present description.
- tortuous plot is meant a non-straight line connecting two points and composed of a series of curves and / or segments oriented in different directions.
- tortuous traces mention may be made in particular of waves of elementary shape such as a sinusoidal, square, triangular or sawtooth shape (see FIG. 17).
- Geometrically less regular plots, without repeated elementary patterns, are also operative.
- the tortuous channel pattern at the diffusion spaces artificially increases the distance between two points of the analysis membrane, or rather increases the temporal distance separating two points of the analysis membrane.
- the present invention has opted for integral analysis membranes whose structure is entirely made of a fiberglass material.
- each reagent in solution in ad hoc adsorption buffer is deposited in its reaction zone where it is retained long enough to be able to adsorb. Forced drying (non-passive, for example by heat treatment and / or by ventilation) makes it possible to further limit the dispersion of the reagents outside the reaction zones.
- Certain embodiments of the diffusion-reduced spaces according to the invention give rise to an effective reduction in the diffusion rate of the liquids.
- the diffusion rate of the water at ambient temperature (of the order of 20-28 ° C.) can in fact be divided by a factor at least equal to 2, with respect to its diffusion rate on the same support of composition based on fiberglass, before modification.
- Such embodiments provide better control over the concentration / amount of reagents finally adsorbed in the reaction zones, and make it possible to increase the contact time between the analytes of the test sample and the reagents present in each zone of the reaction zone. localized reaction in these spaces with slow diffusion.
- the improvement in the quality of the reactions thus obtained is all the more significant for analyzes that require sequential processing of the liquid sample; the reaction zones dedicated to such an analysis are arranged in series, each of the reaction zones being specifically functionalized in order to be able to carry out a particular step of the analysis method.
- an analysis membrane according to the invention requires the liquid to analyze large variations in speed in their diffusion. These large variations in diffusion rate form, just upstream of the spaces with slow diffusion, a bottleneck at which the liquid to be analyzed accumulates. This temporary accumulation makes it possible to reduce the surface area of the liquid exposed to gas exchange and, consequently, to significantly slow down the drying of the liquid to be tested.
- the fiberglass material in this particular part of the analysis membrane, has encrustations of solid wax.
- first intermediate pattern intended to coat the underside of a fiberglass sheet which will constitute the basic structure of the analysis membrane; this first intermediate pattern is in the image of the space diffusion slowed to achieve;
- a second intermediate pattern intended to coat the upper face of the fiberglass sheet; this is an inverted image of the first intermediate pattern;
- the first and second intermediate units may be printed on one and the same transfer film or on two independent transfer films;
- these intermediate units are affixed directly in contact with the two faces of the fiberglass sheet so that, on both sides of the the thickness of this sheet fiberglass, they are found mutually symmetrical, at least substantially opposite one another;
- said fiberglass sheet and the said transfer film (s) are subjected to a heat treatment capable of causing at least partial melting of the wax constituting the intermediate units, and a mechanical treatment able to compress momentarily the thickness of all or part of said fiberglass sheet;
- said fiberglass sheet is subjected to a mechanical and thermal expansion phase, able to allow said sheet to recover at least part of its initial thickness and the wax to resolidify within the thickness of said fiberglass sheet.
- the printing of the intermediate patterns is carried out with a solid ink weakly concentrated in wax and / or in a pale color (weakly intense).
- the intensity of the printed color is proportional to the amount of wax deposited on the transfer film; this intensity of color is chosen so that, once in the thickness of the fiberglass sheet, the amount of wax thus transferred is insufficient to completely block the passage of liquids by capillarity but just sufficient to significantly reduce the speed of this passage.
- this wax impression is implemented using a XEROX ® ColorQube TM type printer, powered with XEROX ® 108R00931 solid reference inks (cyan color), 108R00932 (magenta color), 108R00933 ( yellow color) and 108R00934 / 108R00935 (black color).
- channels or microchannels of small width and / or thin are shaped in the fiberglass material; these micro-channels channel the flow of liquids that pass through said space.
- the average section of these microchannels is advantageously between 10000 ⁇ 2 and 500,000 ⁇ 2 , preferably between 1,000 ⁇ 2 and 150,000 ⁇ 2 , and even more preferably between 30,000 ⁇ 2 and 55,000 ⁇ 2 .
- the channels of the diffusion-delayed spaces have advantageously a width between 150 ⁇ and 1.5 cm, preferably between 200 and 1000 ⁇ .
- a reduction in the diffusion rate of the liquids is obtained thanks to reduced hydrophilicity and / or flow density.
- the diffusion rate of the water (subjected to ambient temperature, that is to say to a temperature of the order of 20-28 ° C.) is divided by a factor at least equal to 2, with respect to its diffusion rate on the same fiberglass composition support, before modification.
- the channels progressing within these spaces comprise at least one portion having a tortuous path.
- the tortuous route of these channels makes it possible to lengthen the fluidic distance that separates two points of the analysis membrane.
- said tortuous trace can be graphically represented as a wave of elementary shape chosen from: a sinusoidal, square, triangular, sawtooth wave or a combination thereof.
- an analysis membrane according to the invention advantageously comprises, localized in a space with slowed diffusion, at least two reaction zones. These reaction zones are arranged in direct fluid communication, one behind the other.
- the reaction zones of the same space with slow diffusion are arranged in indirect fluid communication, one behind the other and between which is interposed at least one buffer zone. Said at least one buffer zone is not functionalized.
- the fiberglass material at this level has not undergone any treatment or any modification in its structure or composition.
- the buffer zones of a space with slow diffusion of an analysis membrane according to the invention have a surface generally of between 0.5 mm 2 and 25 mm 2 , preferably of order of 1-10 mm 2 .
- a width and a length or a base and a height can be defined, then the corresponding widths and lengths or the bases and the corresponding heights, are of the same order of magnitude, usually in a ratio of 1 to 5.
- the distance between two adjacent zones, reaction zones and / or buffer zones is less than 15 mm, preferably less than 5 mm, and more preferentially still is of the order of 1-2 mm.
- the different reaction zones can be functionalised concomitantly with a minimal risk of contamination from one reaction zone to another, and a good control of the amount of reagents charged to each reaction zone.
- a space with slow diffusion of an analysis membrane comprises at least two reaction zones placed in direct fluid communication, one behind the other and at a distance of less than 5 mm, preferably at a distance of less than 5 mm. distance of the order of 1-2 mm.
- An analysis membrane according to the invention may be of solid form.
- the outline of the different deposit zones, reaction zones and channels for communication fluidic is advantageously traced with solid wax.
- the solid wax implanted through the thickness of the fiberglass sheet forms liquid-tight barriers.
- the spaces with slow diffusion advantageously incorporate inlays of solid wax.
- European Patent Application No. EP16163217.9 bioMérieux teaches a method for producing such a solid form analysis membrane.
- An analysis membrane according to the invention may also be of pruned shape.
- said analysis membrane is cut from a sheet of glass fiber material previously coated with a mechanical reinforcing layer on one of its faces, along the outside contour of the hydrophilic network formed by the different deposit zones. reaction, the fluidic conduction channels and the slow diffusion spaces.
- an analysis membrane according to the invention can also be of mixed form.
- it is shaped in a sheet of fiberglass material, one of whose faces, called the lower face, is previously coated with a mechanical reinforcing layer and the contour of the hydrophilic network formed by the deposition and reaction zones. , the fluidic conduction channels and the microchannels of the spaces with slow diffusion, are engraved in the mass of the fiberglass material until reaching the surface of the mechanical reinforcing layer.
- the present invention also relates to a microfluidic device analysis membrane and a microfluidic device comprising such an analysis membrane, characterized, in combination, by all or some of the characteristics above or below.
- FIG. 1 is a schematic representation of a wax impregnation method for forming an analysis membrane according to a first embodiment; in this first embodiment, the analysis membrane is said to be solid and the spaces with slow diffusion are created by wax incrustation;
- Figure 2 shows in (A), a schematic representation of a transfer film used in the wax impregnation process illustrated in the previous figure, and in (B), a photograph of an analysis membrane of solid form obtained from this transfer film;
- Figure 3 Figure 4A and Figure 4B are photographs illustrating the application of a solid form analysis membrane according to the invention for immunodetection purposes;
- Figure 5 is a photograph of an analysis membrane according to the invention shaped for the detection of dengue virus, by a co-detection of the proteins NS 1 and DomII;
- FIG. 6 presents photographs of C0 2 laser etched filter media illustrating the influence of the width of the etching lines on the tightness of the patterns
- FIG. 7 shows a graphical representation (A) and a photograph (B) illustrating the influence of the width of the microchannels on the flow distance of the liquids through a slow diffusion space formed in a fiber filter medium. glass;
- FIG. 8 is a graphical representation showing the correlation between the width of the microchannels and the flow distance of the liquids through a slow diffusion space formed in a fiberglass filter media;
- Figure 9 shows photographs illustrating the influence of (micro) channel width on the flow velocity of liquids through fiberglass filter media;
- Fig. 10 is a graphical representation showing the evolution of liquid flow velocity versus width of (micro) channels formed in fiberglass filter media
- Figure 11 shows photographs illustrating the influence of the thickness of the filter media on the flow of liquids
- Figure 12 is a graphical representation showing the evolution of the liquid flow velocity as a function of the thickness of the filter media
- FIG. 13 is a photographic representation of an example of an analysis membrane according to the invention, shaped by ablation / laser etching, with enlargement of the spaces with slowed diffusion;
- Fig. 14 is a graphical representation illustrating an example of an assay membrane according to the invention shaped by ablation of hydrophilic material and functionalized for molecular detection of target nucleic sequences;
- Figure 15 shows three photographs exposing the results of a molecular detection of a target nucleic sequence, by means of an analysis membrane according to the invention
- FIG. 16 is a photographic representation of another example of an analysis membrane according to the invention, shaped by laser ablation, with enlargement of the spaces with slowed diffusion;
- Figure 17 is a graphical representation of waves of elementary shapes such as sinusoidal, square, triangular and sawtooth forms, tortuous traces in the sense of the present invention may possibly resume.
- FIG. 1 schematically illustrates the implementation of a method for manufacturing an analysis membrane 10, according to a first embodiment of the invention according to which the analysis membrane is of solid form and the Slow diffusion spaces are obtained by wax encrustation.
- an image 12'a corresponding to the pattern to be integrated in the thickness of a fiberglass sheet 11 is created by computer and by means of a drawing software. This first image 12'a is duplicated in a symmetrical image 12'b.
- the two images 12'a and 12'b are printed on a transfer film 20, so as to form two intermediate units 12a and 12b arranged symmetrically with respect to an axis S.
- This axis of symmetry S is also printed on the transfer film 20, as a visual cue.
- Figure 2 (A) illustrates in detail such a transfer film 20 on which are printed the two intermediate units 12'a and 12'b.
- the transfer film 20 is folded in two along the axis of symmetry S, the intermediate units 12a and 12b turned inwards. The latter are thus superimposed on one another.
- a fiberglass sheet 11 is slid inside the folded transfer film 20, interposed between the two intermediate units 12a and 12b.
- the fiberglass sheet 11, sandwiched between the two flaps of the transfer film 20, is then placed in a press between two horizontal, heated compression plates and two rubber parts forming a thermal and mechanical buffer.
- the assembly is subjected to a pressure of the order of 1 kg / cm 2 and at a temperature of 120 ° C for about 3 minutes. During this process, the wax previously printed on the transfer film 20 is transferred onto both sides of the fiberglass sheet 11, and then impregnates the thickness.
- the printing is done with a XEROX ® ColorQube TM type printer, powered with XEROX ® 108R00931 solid reference inks (cyan color), 108R00932 (magenta color), 108R00933 (yellow color) and 108R00934 / 108R00935 (black color).
- the transfer film 20 is an ordinary office paper sheet.
- the intermediate units 12a and 12b printed on the transfer film 20, of generally rectilinear shape, have a flared upper part and a narrow lower part with an open end. Their outer contours are printed in black ink with a high color intensity. In doing so, the amount of wax corresponding has proved sufficient to allow the realization and obtaining impervious edges in the thickness of the sheet of fiberglass.
- the upper part of the intermediate units 12a and 12b is flared and is intended to form a hydrophilic zone, in this case a deposition zone 13a able to receive a liquid sample to be analyzed. Once deposited in the deposition zone 13a, the liquid sample will be able to migrate by capillarity towards the other hydrophilic zones of the device, namely towards the lower part where the reaction zones are located.
- Figure 2 (B) is a photograph of an assay membrane prepared from the transfer film (after special functionalization of the reaction zones and after use).
- This lower part of the pattern corresponds to a space with diffusion slowed according to the invention.
- This space marked in FIG. 2 (B) by a frame with a discontinuous contour, is obtained by transfer and impregnation of a solid ink layer 14 of pale color (weakly intense) applied on both sides of fiberglass sheet. glass 11.
- the intensity of the color to be applied is determined to reduce the hydrophilicity of the fiberglass material without blocking the passage of liquids.
- reaction zones 13b, 13c, 13d and 13e are located within this space with slowed diffusion, spared by the impregnation with wax.
- the position of each of these areas of interest is specifically identified by the marking elements 14b, 14c, 14d and 14e, also drawn in colored solid ink.
- the analysis membrane 10 has been functionalized for the detection of hepatitis B by immuno-detection of the HBs antigen contained in the blood.
- Zone 13d is functionalized by means of anti-HBs monoclonal antibodies specific for the reaction; zone 13d forms the "spot test".
- the 13th zone is functionalized by means of anti-alkaline phosphatase monoclonal antibodies, specific for the detection conjugate; the 13th zone forms the "positive control spot”.
- Area 13c is functionalized with non-specific antibodies to the reaction (e.g., anti-rat antibodies); zone 13c forms the "negative control spot".
- the functionalization of these different zones by the antibodies is carried out by adsorption of the antibodies.
- zone 13b is dedicated to storage of the second part of the conjugate complex (monoclonal antibodies anti-HBs labeled with biotin); zone 13b forms the "anti-HBs-biot Ac spot".
- the deposition zone 13a may also be used for storing the conjugate of the enzyme-linked immunosorbent reaction (streptavidin-alkaline phosphatase or STRE-PAL) in dried form. This conjugate will be resolubilized by the liquid phase of the sample to be analyzed.
- the enzyme-linked immunosorbent reaction streptavidin-alkaline phosphatase or STRE-PAL
- the analysis membrane 10 According to a second mode of application of the analysis membrane 10 previously described, it has been functionalized for the detection, in blood and plasma, of two proteins of the dengue virus: the NSI protein and the domain III of the envelope protein of the virus (DomII).
- zone 13e anti-ALP control zone 0.35 of an anti-alkaline phosphatase antibody 1 mg / ml in PBS,
- test zone 13c 0.35 of an anti-NSI antibody at 1 mg / ml in PBS,
- zone 13d (negative control zone): 0.35 ⁇ M, 0.5% PBS-BSA.
- the analysis membrane is allowed to dry for 3 minutes at 60 ° C. It is then ready for use.
- the underside was previously covered with a liquid impervious mechanical reinforcing layer, in this case an adhesive film made of polyethylene terephthalate.
- a series of seven circular patterns has been laser engraved with a sufficient depth of etching to reach the mechanical reinforcing layer.
- the engraved circles have the same internal diameter of 10 mm, while their engraved edges are of varying thickness, ranging from 0.2 mm to 1.4 mm, in leaps of 0.2 mm.
- a drop of dye is deposited in the center of the patterns to check their tightness.
- Table 1 presents the characteristics of the filter media tested as well as the operating parameters applied to the C0 2 laser used.
- a series of patterns comprising a reservoir R linked to a zone of slow diffusion Z, comprising a micro-channel of variable width from one pattern to another, have been etched by laser C0 2 .
- the widths tested are 80, 100, 120, 140 and 160 ⁇ .
- a constant volume of dye (20 ⁇ ) is then deposited in the reservoir zone. The distance traveled by the dye in the channel is measured in each case. The results are presented numerically in Table 2 below and graphically in Figure 8. Width of microDistance
- the distance (d) traveled by the dye in the microchannels is proportional to the width (C) thereof. It is thus possible to control the routing of liquids by means of the laser engraving method described here. Moreover, the implementation of such an analysis makes it possible, for a given fiberglass material, to evaluate the width of the microchannels and the distance between two reaction zones / buffer to be applied to a space with slow diffusion. of an analysis membrane according to the invention. c) Determination of the influence of the (micro) channel width on the flow velocity of liquids
- the nominal migration rate of a liquid of viscosity close to water in the Whatman TM MF1 type fiberglass support (367 ⁇ thick) is of the order of 2 mm / s.
- fiberglass membranes the underside of which is reinforced with a PET film, have been shaped by the C0 2 laser, by ablation of material in order to emerge (micro) channels of different widths.
- FIG. 9 shows three of the membranes thus prepared, denoted A, B and C. These membranes have a central zone O in which the colored liquid has been deposited, and the diffusion of this liquid through (micro) channels 6A, or 6c, arranged on either side of the central zone O, was followed.
- the width of the micro-channel 6A of the membrane A is 100 ⁇ , that of the microchannels of the membrane B, 400 ⁇ , and that of the channels C of the membrane C, 3 mm.
- fiberglass membranes the underside of which is reinforced with a PET film, have been shaped with the C0 2 laser, by ablation of material so as to create zones of lesser thickness.
- the application of a laser makes it possible to eliminate a given percentage of the thickness of the material, this thickness varying with the power and the speed of the laser. If the power / speed ratio is too high, the etching is too deep and tends towards the total elimination of the material. If the power / speed ratio is too low, the impact of the etching on the confinement of the liquids is negligible.
- ⁇ residual thickness of the membrane
- the etched areas SD of the membrane D have a residual thickness of the order of 120 ⁇ .
- the residual thickness of the etched areas SE of the membrane E is of the order of 170 ⁇ . These residual thicknesses of paper on the etched areas were measured using a comparator, a mechanical instrument for determining very small thicknesses.
- the maximum thickness of paper type MF1 for effective retention of liquids, that is to say a decrease of half the speed of liquids within it, is around 130 ⁇ .
- the exemplified analysis membrane comprises two hydrophilic analysis networks arranged head-to-tail, each hydrophilic network comprising:
- a space with slow diffusion containing three crossing reaction zones 32a or 32b, 33a or 33b, and 34a or 34b, aligned one behind the other; the distance between two adjacent reaction zones is of the order of 1 mm.
- a secondary deposition or secondary storage zone, 35a or 35b placed in direct fluid communication with the reaction zone 33a or 34b, through a micro-channel.
- each reaction zone is in direct fluid communication with the adjacent reaction zone, through a single conical micro-channel.
- each reaction zone is in direct fluid communication with the adjacent reaction zone, through three parallel microchannels and of generally rectilinear shape.
- Figure 14 shows an etching pattern according to a particular configuration of an analysis membrane 40 according to the invention, shaped in Whatman TM MF1 type fiberglass material, for detection of target nucleic sequences.
- the corresponding analysis membrane 40 comprises, upstream, a mixed zone 37, in that it serves, firstly, to store in advance the dry reagents and, secondly, to receive the liquid reagents ( sample and enzymatic substrate) during the analysis.
- a slow diffusion space in which is located a series of reaction zones 38a-38d, positioned as scale bars and interconnected by micro-channels. These micro-channels have a beveled shape, with a maximum width of the order of 300 ⁇ .
- the third zone 39 at the outlet of the space with slow diffusion, serves as a capillary pump for the flow of liquids.
- the channel which ensures the fluidic communication between the zone 37 and the reaction zones of the space with slowed diffusion merges structurally with a part of the zone 37 and constitutes an extension thereof auditing space slowed down.
- the zone 37 is functionalized by drying the following detection reagents, in a buffer which allows their easy redissolution by the sample: i) oligonucleotides, specific for the single-stranded DNA target to be detected, labeled with biotin ii) oligonucleotides, specific to another part of the DNA target sequence, labeled with horseradish peroxidase and iii) alkaline phosphatase labeled streptavidin.
- the reaction zones 38a-38e serve for the specific capture and the revelation of the presence of the target DNA.
- the first reaction zone 38a is functionalized by adsorption of monoclonal antibodies directed against horseradish peroxidase (specific test).
- the following three reaction zones 38b, 38c and 38d are functionalized by adsorption of BSA. They serve here as a negative control but could also allow multiplexed detections.
- the last reaction zone 38e is functionalized by adsorption of BSA-biotin (positive control).
- Figure 15 illustrates the results of a detection using such an analysis membrane. From left to right, the target nucleic acid concentrations are 0 (negative control), 1 nM and 10 nM.
- the analysis membrane 50 was shaped into Whatman TM MF1 fiberglass material by C0 2 laser ablation cutting.
- the analysis membrane 50 comprises:
- zone 59 at the outlet of the space with slow diffusion, which serves as a capillary pump for the flow of liquids.
- the slow diffusion space here corresponds to a conduction channel portion at which the channel has a width of the order of 1.0-1.5 mm and a tortuous path.
- This plot has the shape of a triangular elemental wave.
- the passage time of the liquids through the different reaction zones 58a, 58b and 58c is thus regulated, on the one hand, by the small width of the channel and, on the other hand, by its tortuous pattern.
- the analysis membrane 50 is covered with a transparent adhesive film, applied on its underside.
- the assembly is surmounted by a rigid polymer plate (of the order of 1 mm thick).
- this plate is transparent and is made of polymethylmethacrylate, or PMMA.
- the underlying analysis membrane thus remains visible in its entirety, facilitating visual tracking of the diffusion of the liquid to be analyzed through the analysis membrane.
- the analysis membrane is confined in a housing made of an opaque polymer material.
- the upper face of this housing may possibly take up the configuration of the PPMA transparent plate, which has cut-out openings positioned in relation to the areas of interest of the analysis membrane, in particular the zone for depositing the sample 57. and the reaction zones 58a, 58b and 58c.
- reaction zone 58b corresponds to a test zone
- reaction zone 58a the reaction zone 58a, at a negative control zone
- reaction zone 58c the reaction zone 58c, to a positive control zone.
- the part (a) shows the visual of a test to the negative result and the part (b), the visual of a test to the positive result.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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EP2016163345 | 2016-03-31 | ||
PCT/EP2017/057512 WO2017167861A1 (fr) | 2016-03-31 | 2017-03-30 | Membranes d'analyse de dispositifs microfluidiques, réalisées en un matériau en fibre de verre |
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EP3436194A1 true EP3436194A1 (fr) | 2019-02-06 |
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EP17713317.0A Withdrawn EP3436194A1 (fr) | 2016-03-31 | 2017-03-30 | Membranes d'analyse de dispositifs microfluidiques, réalisées en un matériau en fibre de verre |
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