• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Functionalized porous microchips and their use in the development of sensors. The present invention relates to microchips with a printed circuit on its surface formed by a communicating inlet and outlet mouth and with a porous material contained within this circuit that has been obtained by the technique of evaporation of the solvent (breath figures) and in whose pores contains active molecules anchored through interactions of the host-guest type. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2676668A1
    申请号:ES201631683
    申请日:2016-12-23
    公开日:2018-07-23
    发明作者:Juan RODRIGUEZ HERNANDEZ；Alberto Gallardo Ruiz；Alfonso FERNÁNDEZ-MAYORALAS；Agatha Bastida Codina
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    5 The present invention relates to microchips with a printed circuit on their surfaceformed by an inlet and outlet outlet communicated and with a materialporous content within this circuit that has been obtained by the technique ofevaporation of the solvent (breath figures) and in whose pores it contains moleculesassets anchored through host-guest interactions. STATE OF THE TECHNIQUE
    The microdevices have increased their presence on a daily basis, especially in the form of electronic microdevices and micro-electro-mechanical sensors,
    15 as part of everyday objects such as mobile phones, toys, appliances or cars. The success of this type of devices is due to factors such as its response speed, its compact design, the possibility of being produced in series at reasonable prices and the low energy consumption derived from its use.
    20 Within this miniaturization process, microfluidics (in English microfluidics), has been responsible for understanding how fluids can be manipulated and analyzed on a micrometric scale, thus reducing significantly the volume of the samples used. Subsequently, they began to develop microfluidic systems
    25 capable of integrating functions into the chip itself. These systems, which are known by the term "lab on a chip" ("Lab on a chip" or "MicroTAS", TAS = total analysis system), allow the incorporation of different functionalities within a single microfluidic device, which facilitates the execution of several chain processes. As a result, they are being employed in a wide range of
    30 applications, especially in the field of biochemistry and biomedicine, having served, among others, as a tool for cell analysis, tissue incubation or genome studies.
    However, despite the advantages of “lab on a chip” devices, the
    The development of this type of systems still requires: on the one hand, increase the versatility of the production processes used in its manufacture (its manufacture


    currently uses expensive processes such as lithography); on the other hand, they require new processes of functionalization of the microchannels that allow to carry out the desired processes such as reactions, selective immobilizations or recognition processes.
    5 In recent years, sophisticated methods for obtaining porous and functionalized surfaces have been developed. Within these, the solvent evaporation technique (breath figures in English), is an interesting, low-cost and simple alternative that allows the design of porous materials that can be controlled
    10 pore size. Pore formation in this technique occurs when a polymer solution is maintained in a volatile solvent within a closed chamber with high relative humidity. During evaporation of the solvent, the solvent / air interface cools and micrometric drops of water condense on the surface. Finally, the evaporation of the water droplets deposited leads to the
    15 formation of a porous film that presents a hexagonal arrangement simulating the pattern of a honeycomb. The paper by Rodríguez-Hernández et al (Journal of Colloid and Interface Science 457 (2015) 272-280) describes the formation of a polymeric material modified from styrene modified with cyclodextrin obtained by the solvent evaporation technique, which is functionalized with polyacrylate modified with
    20 adamantane linked by host-guest interactions on cyclodextrins anchored in the pores. In the document ACS Appl. Mater. Interfaces 2015, 7, 12210-12219, describes the obtaining of enzymatic catalysts obtained by the solvent evaporation technique that are functionalized by the non-covalent anchoring of the ALP enzyme to the pores.
    25 The functionalization of porous materials in general and in particular of this type of materials within a microfluidic device has been treated rarely. In most cases the treatment of microfluidic devices requires the use of particular methods of modification normally using
    30 more than one stage. Thus, surface activation processes using UV light or through ozone treatments are required to create functional groups in which to immobilize the desired molecule. In addition, the location or control of the density of immobilized groups is difficult to control. The density of groups can also be varied depending on the amount of functional polymer.
    35 incorporated in the initial mixture.

    DESCRIPTION OF THE INVENTION
    The inventors have developed a strategy for the preparation of porous materials using two components: one is the matrix and the other a copolymer that
    5 has the desired functional groups. This strategy allows:- Density control of functional groups-Selective modification inside and / or outside the pore-Use of little functionalized copolymer (more expensive than the matrix)-Possibility of having more than one functional group within the pore
    10 -A porous channel implies a greater surface area and a better one: catalytic activity, response to detection etc. depending on the end use we give it.
    Other advantages of this method are: the rapidity (the pores form in less than one
    15 minutes), cost (no expensive equipment, only a wet chamber), modularity (treatment of all types of organic, inorganic substrates), versatility (a chip could be made in which the detector part (porous) can be easily interchangeable depending on the molecule to be detected or the reaction to be carried out)
    Therefore, in a first aspect, the present invention relates to a microchip comprising:
    A. a support comprising a printed circuit on its surface formed by
    at least one inlet and at least one outlet communicated by a channel that in turn contains at least one reservoir and
    B. a functionalized porous material incorporated into the printed circuit and obtained by the solvent evaporation technique on the same support, comprising at least one type of active molecule immobilized inside
    30 of the pores by either covalent or non-covalent interactions, preferably host-guest interactions.
    In the present invention, interactions of host-guest type forces between molecules or ions other than covalent bonds, such as bonds of
    35 hydrogen, ionic bonds, Van der Waals forces or hydrophobic interactions that occur specifically between two molecules. Host-guest interactions


    they can form and break reversibly depending on the conditions of the environment, the presence of other molecules with higher affinity or varying the concentration of the host molecule, among other factors.
    5 In a preferred embodiment, the matrix of the porous material is formed frompolystyrene, polyacrylates, polycaprolactones or lactic polyacid.
    In another preferred embodiment, the pores of the porous material contain immobilized groups obtained through the introduction of an additive (additive: copolymer
    10 block or not containing the desired functional groups in its structure) which are selected from cyclodextrins or their derivatives, amino groups or their derivatives, alcohol groups or their derivatives, or carboxylic acid groups or their derivatives. In a more preferred embodiment, the immobilized groups are cyclodextrins.
    In another preferred embodiment, the active molecule immobilized inside the pores is selected from enzymes, nucleic acids, antibodies, adhesion proteins, fluorophores, chromophores or sensing molecules. In a more preferred embodiment, the immobilized molecule is the HRP enzyme.
    In another preferred embodiment, the active molecule is modified by the addition of a chemical group, preferably adamantane groups that can be selectively and non-covalently immobilized on surfaces containing cyclodextrin groups.
    In a more preferred embodiment, the active molecule is an enzyme modified with adamantane groups. The modification of enzymes and proteins among others is delicate since its secondary structure can be affected and therefore its functionality. In this case the modification with adamantane does not interfere in the structure of the enzyme keeping its catalytic activity.
    In another preferred embodiment, the support is glass, silicon, or polymeric. This support must be transparent for detection by UV-vis or fluorescence and / or conductor for evaluation of the sensing capacity by electrochemical methods.


    Another aspect of the invention relates to the use of the microchip as described above for the detection of substances, for carrying out reactions (for example electrochemical) or for enzymatic catalysis.
    5 In most tests the detection is done visually, either by UV spectroscopy or fluorescence. Another possibility is to have a conductive matrix polymer and the support also so that detection could be done, for example, by electrochemical methods.
    Another aspect of the invention relates to a sensor comprising the microchip as described above.
    To obtain the microchips of the present invention, the technique of evaporating the solvents can be carried out in two ways:
    15 i. introducing the microchip into a humid chamber and depositing the solution to evaporate inside the chip wells. So once the solvent evaporates the chip will have the wells with functionalized pores, or
    ii. using a closed device that is filled with the solution. After introducing moist air into the device that empties the channels but leaves
    20 solution-filled reservoirs. This air is allowed to pass during the evaporation process so that pores are also formed in the cavities.
    Therefore, another aspect of the invention relates to a method of obtaining a microchip as described above comprising at least
    following steps: a) obtain a solution of the precursors of the porous material in at least one volatile organic solvent, b) deposit the solution of the previous stage in the printed circuit channel
    30 of the support, c) passing a flow of air with a relative humidity greater than 90% over the channel surface, or allowing it to evaporate in an atmosphere saturated with water vapor, and d) adding the active molecule on the porous material obtained in The phase
    35 above.


    In a preferred embodiment, the volatile organic solvent used in step (a) is selected from THF, CS2, acetone, CHCl3 and any combination thereof. More preferably the volatile organic solvent used in step (a) is selected from THF, CHCl3 and any combination thereof, more
    5 preferably still the solvent comprises THF and CHCl3, being obtained with saidmix a surface with a more homogeneous pore distribution.
    In another preferred embodiment, the porous material precursors of step (a) are unmodified or modified styrene monomers, acrylate monomers without
    10 modify or modified or mixtures thereof. In a more preferred embodiment, the precursors of the porous material are styrene monomers modified with cyclodextrin.
    In another preferred embodiment, the active molecule is selected from enzymes, nucleic acids, antibodies, adhesion proteins, fluorophores, chromophores or sensing molecules.
    In another preferred embodiment, the active molecule is modified by the addition of a chemical group.
    In a more preferred embodiment, the active molecule is an enzyme modified with adamantane groups.
    The advantages of the invention over other known strategies is that they can be
    25 immobilize other types of molecules: protein recognition sequences, or cell adhesion sequences for example. The immobilization can be covalent or non-covalent. In the latter case, the original substrate can be recovered and reused. The first one gains in stability and durability of the device. Both can be done by the solvent evaporation technique of the invention.
    Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the
    Invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

    BRIEF DESCRIPTION OF THE FIGURES
    FIG. 1 Operation scheme of a stereolithography equipment.
    FIG. 2 a) Scheme of the structure of the device used for the functionalization of the device by controlled flow; b) Section view of the device assembly.
    FIG. 3 a) Hermetic working chamber. b) Images obtained in SEM of the porous structures obtained for a mixture of PS and PS-b-PDMAEMA.
    FIG. 4. Scheme of the method of formation of porous structures in chip reservoirs.
    FIG. 5. a) ABTS oxidation scheme by interaction with HRP and H2O2. b) reaction control. c) green color observed as a result of ABTS oxidation. d) soluble HRP activity, HRP-Ada and its control.
    FIG. 6. Immobilization of a) unmodified HRP on the surface of HPS + P (S-co-SCD); b) HRP-Ada on the surface of HPS and c) HRP-Ada on the surface of HPS + P (S-co-SCD). In all cases the enzyme was incubated for 60 minutes with a concentration of 0.01 mg / ml in 40 mM PB buffer pH 6.8. d) Variation of absorbance at 405 nm during catalytic oxidation of ABTS using substrates a) -c). e) Activity of the different substrates.
    FIG. 7. a) Evolution of absorbance measured during the first three catalytic cycles; b) Enzymatic activity measured during 5 consecutive cycles evidencing a constant surface activity.
    FIG. 8. a) Evolution of absorbance at 405nm for control surfaces, surfaces with HRP in the first cycle and upon removal from the surface using β-CD in solution. b) Catalytic activity of the surfaces explored in a).

    EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention. 5
    1. Design of the support with the printed circuit: three-dimensional modeling and 3D printing.
    The virtual 3D models from which the microchip parts were printed
    10 of the invention were generated with computer-aided design software or CAD (acronym for computer aided design). This format divides the virtual model into successive two-dimensional cross layers based on the resolution of the printer. Finally, the printer generates the volume by adding material in each of these layers. The printer used was a desktop computer with
    15 MicroSLA technology (stereolithography). Print a maximum volume of 43x27x150mm per 30 μm layers with an X-Y resolution of 165 μm. Stereolithography is an additive manufacturing technology that manages to generate volumes solidifying layer by layer photopolymerizable resins after irradiation with UV light. In this process, the piece is printed on a platform
    20 horizontal, submerged in said resin. Thanks to high-precision mirrors it is possible to focus the laser beam on the surface of the resin, so that the irradiated material solidifies on the printing platform, creating layers whose thickness depends on the resolution of the printer, generally oscillating between
    0.015 and 0.15 mm (Fig. 1). Once a layer is irradiated, the printing platform is
    25 shifted to allow solidification of a new layer of material over those already generated. This process was repeated successively until the final volume was obtained. Once printed, the pieces were immersed in ethanol to remove excess resin with which they leave the printer and subsequently underwent a post-curing process (irradiation of the piece with UV light) to complete the photopolymerization.
    2. Incorporation of porous material into the microchip
    To obtain functional microchips, once the support was obtained by 3D printing, the porous material was incorporated into this support. As shown schematically in Fig. 2, the printed piece will have an inlet and an outlet, communicated by a channel through which it is circulated


    a fluid Reservoirs of different size, shape and number were placed along the canal where both hydrogels and porous materials were introduced. Thus, once the device is manufactured, the liquid of interest will circulate through it and will come into contact with the material previously encapsulated in the reservoirs of the
    5 device
    To obtain the porous material, four different polymers were used to study both its immobilization in the chip and the formation of pore in the wells of the printed pieces. The structure of the polymers used is as follows:
    (i) Polystyrene-b-poly (N, N'-dimethylaminoethyl methacrylate) (PS-b-PDMAEMA), (ii) functionalized polystyrene-mono amino (PS-NH2), (iii) polystyrene-b-poly (acrylic acid ) (PS-b-PAA) and (iv) polystyrene and polystyrene statistical copolymer modified with modificado-cyclodextrin (-CD).


    These polymers were previously synthesized. For the formation of the porous structures each of these polymers was mixed with polystyrene (PS) of high molecular weight (250 kDa) in a proportion of 20 to 80 by weight, that is, with PS as a major component and dissolved in chloroform with a concentration in polymer 5 of 30mg / ml. The proposed systems will allow porous structures to be formed with a chemically distinct pore interior outside the pore. The functional groups will tend to be part of the interior of the pore giving rise to structures having dimethylamino groups, primary amines, acid groups or cyclodextrins respectively.
    10 Two methods have been used for the deposition of the materials previously mentioned in the printed chips:
    a) Direct deposition in reservoirs.The first series of experiments was carried out by injection so
    15 manual of the different components inside the device. For this purpose, a micropipette was used with which an exact amount can be deposited in each reservoir manually in a chamber with a controlled relative humidity. The specific case of the deposition of solutions for the formation of porous surface structures must be carried out under conditions of relative humidity.
    20 greater than 90%. To achieve these working conditions, an airtight chamber was used that has allowed to achieve and maintain this moisture condition. The depositions were made on printed pieces with three reservoirs 4mm in diameter and 0.25mm deep. 7 μL of the different PS-polymer solutions have been deposited on each reservoir. The following images obtained
    25 by SEM show the formation of porous films inside the reservoirs (Fig. 3).
    b) Formation of the pores in the device by controlled flow of the components.
    30 The second method of forming porous structures within the device was the injection of the various precursors using a precision peristaltic pump (Fig. 4). The device is composed of the microchip that will be covered with a lid to make it waterproof (typically a transparent silicone sheet). The chip contains an input and an output with a certain number of reservoirs (in this
    35 case was varied between 2 and 4) with diameters (between 200 microns and 2mm) and variable depths (from 100 microns to 3mm). The reservoirs, deeper than the canal


    that unites them, will allow to form functionalized porous structures. The process is done in two stages. In the first one, the solution containing the polymer mixture is circulated in the circuit. In the second stage, moist air that cleans the dissolution channels and allows the pore formation process to pass through
    5 do in the reservoirs that, these yes, are full of the solution.
    3. Functionalization of porous surfaces
    a) Immobilization of biomolecules and / or sensor molecules.
    The chips of the invention described herein have been prepared with the monomeric precursor (i) mentioned above with pores containing poly (dimethylamino ethylmethacrylate) groups. These functional groups are capable, for example, of forming colored complexes when they react with nitro groups (contained for example in trinitrotoluene (TNT) by the Meisenheimer reaction).
    15 b) Immobilization of the enzyme inside the pores. Before the immobilization step, the enzyme was first partially modified with adamantane (Ada) groups following previously described procedures. The strategy involves the reaction between the amino acid amine groups of
    20 lysine present on the surface of the enzyme and carboxylic acid groups of a modified adamantane molecule. This strategy allows the enzyme to be functionalized while maintaining the secondary structure and its catalytic activity. Subsequently, the films were immersed in 0.32 ml of a solution of HRP-Ada (0.01 mg / ml) at 40 mM pH 6.8 of potassium phosphate for 4 h. In moments
    At different levels, the supernatant solution was measured at 595 nm with the Protein (Bio Rad) reagent assay to determine the concentration of non-adsorbed protein. The films were then rinsed with water, 40 mM potassium phosphate pH 6.8 and 40 mm potassium phosphate with 0.25% bovine serum albumin and 0.5% Triton X-100 (v / v) at 25º. As a control, blank experiments were carried out.
    30 using HPS surfaces without -CD residues and unmodified HRP enzyme (0.01 mg / ml) to assess the presence of non-specific interactions.
    4. Enzymatic activity within the microchip
    35 To determine the catalytic activity of the immobilized enzyme, the reaction in which HRP is involved is studied. This enzyme binds to H2O2 forming


    a complex capable of oxidizing different compounds. In this case, azidobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as the model substrate. Thiscompound, in its oxidized form, has a bluish green color, which can beEasily detect by measuring UV-Vis at a fixed wavelength of λ = 405 nm. For5 test the effect of enzyme modification with Ada, activity was exploredcatalytic solution of the unmodified enzyme (HRP), the enzyme that carries residuesof adamantane (HRP-Ada) and a control experiment without H2O2 (Control). TheExperiments (shown in Figure 5) revealed that the activity in solution of theHRP after modification with adamantane decreased while maintaining, however,
    10 about 50% of the initial activity.
    In order to understand the kinetics of the modified surfaces, the evolution of absorbance was measured at different times for the samples represented in Figure 6. Figure 6d shows the evolution of absorbance as a function of reaction time. A notable and linear increase in absorbance was only obtained in the case of Ada modified HRP on P (S-co-SCD) surfaces. As seen in the figure, only in this case the deposit where the catalysis occurs becomes green after 1 h of incubation. In the rest of the cases an almost negligible catalytic activity can be observed. This residual catalytic activity can
    20 be attributed to the chemical hydrolysis of the substrate which, as expected, can be neglected when compared to enzymatic hydrolysis in the case where host-host interaction occurs. The HRP-Ada activity supported was ~ 33 U / mg (Figure 6e).
    25 5. Stability of enzymatic immobilization and recovery evaluation.
    Once the specificity of the host-guest interaction to immobilize only the enzymes carrying Ada groups was demonstrated, the stability of these interactions and the enzymatic activity during the repetitive cycles were explored. The use of host interactions
    30 guest allows enzymatic recovery using the appropriate conditions indicated below.
    To determine whether surfaces and in particular host-guest interactions are strong enough to carry out several reaction cycles, the
    The same catalytic test on the same surface for five cycles. After each cycle, the surface was thoroughly washed with PB buffer. How


    it can be seen in figure 7a, the surfaces show catalytic activity during the explored cycles (up to five). More interestingly, the observed catalytic rate is stabilized during the cycles and exhibits an average enzymatic activity of 28 ± 5 U / mg.
    5 In view of the potential use of these catalytic surfaces with other enzymes and taking advantage of host-guest interactions to reuse these porous functional surfaces, the possibility of recovering the immobilized HRP-Ada enzyme was evaluated. For this purpose, a displacement of the immobilized HRP-Ada was attempted from the
    10 surface by an excess of free β-CD. Surfaces containing HRP-Ada were immersed in an aqueous solution of 6.0 mg / ml of β-CD in phosphate buffer pH = 6.8 with 0.2% Triton X100 and 3 mg / ml of albumin for 2 ha 25 ° C The supernatant was removed and then the surfaces were washed several times. Subsequently, the catalytic test was repeated using the same conditions.
    As shown in Figure 8a, no catalytic activity could be observed on surfaces exposed to β-CD. Therefore, it can be concluded that the release of HRP-Ada from the substrate has been achieved successfully (Figure 8b). In addition, it can be estimated, taking into account the activity of the released enzyme, that approximately 1-3 μg of HRP enzyme were adsorbed at about 1 cm 2.

    权利要求:
    Claims (17)
    [1]
    1. Microchip comprising:
    A. a support comprising a printed circuit on its surface formed by
    5 at least one inlet and at least one outlet communicated bya channel that in turn contains at least one reservoir and
    B. a functionalized porous material incorporated into the printed circuit and obtained by the solvent evaporation technique on the same support, comprising at least one type of immobilized active molecule
    10 inside the pores through covalent or non-covalent interactions of the host-guest type.
    [2]
    2. Microchip according to claim 1, wherein host-guest interactions do not
    Covalent are hydrogen bonds, ionic bonds, Van der 15 Waals forces or hydrophobic interactions.
    [3]
    3. Microchip according to any one of claims 1 or 2, wherein the matrix of the porous material is formed from polystyrene, polyacrylates, polycaprolactones or lactic polyacid.
    [4]
    4. Microchip according to any one of claims 1 to 3 wherein the pores of the porous material contain immobilized groups that are selected from cyclodextrins or their derivatives, alcohol groups or their derivatives, amino groups or their derivatives, or carboxylic acid groups or their derivatives.
    [5]
    5. Microchip according to any one of claims 1 to 4 wherein the active molecule immobilized inside the pores is selected from enzymes, nucleic acids, antibodies, adhesion proteins, fluorophores, chromophores or sensing molecules.
    [6]
    6. Microchip according to the preceding claim wherein the active molecule is modified by the addition of a chemical group.
    [7]
    7. Microchip according to any of claims 5 or 6 wherein the active molecule is an enzyme modified with adamantane groups.

    [8]
    8. Microchip according to any one of claims 1 to 7 wherein the support is made of glass, silicon or polymeric.
    [9]
    9. Use of the microchip according to any of claims 1 to 8 for the detection of substances or for enzymatic catalysis.
    [10]
    10. A sensor comprising the microchip according to any one of claims 1 to
    [8]
    8.
    11. Method for obtaining a microchip according to any one of claims 1 to 8 comprising at least the following steps: a) obtaining a solution of the precursors of the porous material in at least one volatile organic solvent, b) depositing the solution of the previous stage in the channel of the printed circuit 15 of the support, c) passing an air flow with a relative humidity greater than 90% on the channel surface, and d) adding the active molecule on the porous material obtained in the previous stage . twenty
    [12]
    12. Process according to the preceding claim wherein the volatile organic solvent is selected from THF, CHCl3 and any combination thereof.
    [13]
    13. Method according to any of claims 11 or 12 wherein
    Porous material precursors are unmodified or modified styrene monomers, unmodified or modified acrylate monomers or mixtures thereof.
    [14]
    14. Method according to the preceding claim wherein the styrene monomers 30 are modified with cyclodextrin.
    [15]
    15. Method according to any of claims 11 to 14 wherein the active molecule is selected from enzymes, nucleic acids, antibodies, adhesion proteins, fluorophores, chromophores or sensing molecules.

    [16]
    16. Method according to the preceding claim wherein the active molecule is modified by the addition of a chemical group.
    [17]
    17. Method according to the preceding claim wherein the active molecule is an enzyme modified with adamantane groups.

    DRAWINGS
    FIG. 1 a) b)
    FIG. 2

    b)
    to)
    FIG. 3
    FIG. 4

    FIG. 5

    FIG. 6

    FIG. 6 (CONT.)
    a) b)
     Control
     Cycle 1
    1.2
    A (U / mg)
    30
    twenty
    10
     Cycle 2 Cycle 3
    0.8
    0.4
    0.0
     0 20 40 60 80 Time (min)
    FIG. 7
    OD (405 nm)

     Control (b)
     (a) 1.5
     Cycle 1 30
    A (U / mg)
     After desorption HRP-Ada removal
    1.2
    twenty
    0.9
    0.6
    10
    0.3
    0
    0.0
     0 20 40 60 80 Time (min)
    FIG. 8
    OD (405 nm)
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    同族专利:
    公开号 | 公开日
    ES2676668B1|2019-05-20|
    WO2018115558A1|2018-06-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB201403790D0|2014-03-04|2014-04-16|Ge Healthcare Ltd|Microfluidic devices and arrangements for introducing reagents and biological samples to such devices|
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    PCT/ES2017/070831| WO2018115558A1|2016-12-23|2017-12-20|Functionalised porous microchips and use thereof in the production of sensors|
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