Microfluidic device

Abstract

The invention generally relates to microfluidic devices and uses thereof. In particular, the present invention relates to a microfluidic device and its use for analyzing or collecting root exudates and / or root microbiome exudates both within different regions of the root as well as at different moments.

Description

[0001] MICROFLUIDIC DEVICE

[0002] FIELD OF THE INVENTION

[0003] The invention generally relates to microfluidic devices and uses thereof. In particular, the present invention relates to a microfluidic device and its use for analyzing or collecting root exudates and / or root microbiome exudates both within different regions of the root as well as at different moments.

[0004] BACKGROUND OF THE INVENTION

[0005] Roots exude biomolecules that modify the physical, chemical and biological properties of their environment. For example, root exudates increase the ability of soils to retain water but also reduce the surface tension, which is an important characteristic for the rewetting of dry soil. Root exudates contains organic acids that reduces the soil pH, contain enzyme that break down organic materials and contribute to the mineralisation of nutrients and facilitate the solubilisation and transport of mineral element to the plant. Root exudates also contain diverse polysaccharides and compounds with microbial and antimicrobial properties such as phenols, peptides, enzymes, amino acids, nucleotides, hormones, organic acids, fatty acids. These compounds in turn modify significantly microbial communities in the rhizosphere.

[0006] Exudates can be extracted from plants grown in the field, but this requires excavation and washing of the root system. Protocols usually consist of applying gentle wash and several purification steps before the samples can be analysed using conventional analytical techniques such as HPLC or mass spectrometry. To avoid excavation of the root, microdialysis probes have been used to measure the flow of exudates continuously, but measurement made using such systems are local and can be difficult to apply to a soil. To overcome the difficulty of working with soil, it is possible to sample the solution from plants grown in hydroponic or semi hydroponic systems. The exudates are then purified from the solution extracted. Exudate extraction in hydroponic systems is easier but may not correlate well to conditions in soil because of water saturation. Soil as growth substrate offers a more natural environment for the root, but the sample extraction from the hydroponic system is easier. Nonetheless, these methods are very labour active, time consuming and require trained personnel. Moreover, they cannot give detailed information about the spatial and temporal properties of exudation processes and cannot give any information about an individual plant. The above mentioned methods provide bulk information, only.

[0007] For the deeper understanding of root exudation and root-microbe interactions it is essential to know the exudation profile along the root, in space and in time as well. Using artificial soils combined with Fluorescent Light Sheet Microscopy can reveal the rootmicrobe dynamics or biological activities in the close proximity of the root but it cannot provide any information about the chemicals involved in this process.

[0008] Recently, microfluidic techniques have emerged as a powerful approach to observe and measure rhizosphere processes. Microfluidics approaches have enabled live observation of root interactions with microbes. Artificial soils can also be used to control the physical structure of the substrate while enabling live observation within the chip. Microfabrication techniques can be employed to mimic the structure of soil and the mapping of simple chemical changes like pH within the rhizosphere has been achieved but the detection of specific molecules remain challenging. The fabrication of microfluidic chips often requires specialised laboratories. Access to clean room, spin coater and plasma treatments machines, photolithography, soft lithography and ion beam lithography, dry and wet etching. Knowledge of materials science and chemistry is also required.

[0009] Microfluidics also offers the possibility to investigate root-microbe interactions and it has the advantage of high-throughput measurements, and using transparent materials it possible to investigate them with optical microscopes. Microchannels can also be used to influence a specific part of the root, however, due to diffusion, it is difficult or even impossible to extract samples from a well-defined root segment.

[0010] There are some methods for the analysis of exudates in specific root segments, such as the methods described in Marschner, H. et al., (Physiologia Plantarum, 1987, 71 , 157- 162) or in Neumann, G. & Rdmheld, V.,. Plant and Soil, 1999, 211 , 121-130). In the work of Marschner et al., after a period of incubation time, the roots are covered with a thin layer of agarose. After a period of 4 h, the agarose is cut into segments 2-3 cm long and their contents analyzed. Neumann et al., disclose a method in which the root systems are spread over the surface of a wet fleece covered with a layer of wet filter paper. Small sheets of polyethylene were placed under the root zones of interest and the root zones were incubated between small pieces of wet filter paper. The filter pieces used for exudate sampling were removed after 3 h for further analysis. Both methods require the destruction of the root to do the analysis.

[0011] Therefore, there is a need of new systems that allows the analysis of exudates from specific segments of the root, avoiding mixing between different root areas without violating the root growth or root biological properties.

[0012] SUMMARY OF THE INVENTION The authors of the present invention have developed a wax printed pattern based microfluidic device for the investigation of plant root exudates spatially and temporally. The applied wax patterns can separate different areas of the root system without violating the root growth or its biological properties.

[0013] Thus, in a first aspect the present invention relates to a microfluidic device, hereinafter the microfluidic device of the invention, comprising: i) a slide of an elastomeric material ; ii) a substrate sheet which contains a pattern of substantially parallel channels, wherein the channels in the pattern are defined by the space between regions in which a hydrophobic material has been deposited onto the substrate; and iii) a backing slide, wherein the substrate sheet is placed between the slide of elastomeric material and the backing slide, wherein the slide of elastomeric material and / or the backing slide is transparent, and wherein the backing slide and the slide of the elastomeric material are bonded to each other at those regions in which the substrate sheet does not separate the two surfaces thereby creating an inlet and an outlet at the opposite ends of each channel in the device.

[0014] In another aspect, the present invention relates to a method for analysing or collecting root exudates and / or root microbiome exudates from different regions within the longitudinal axis of a root, the method comprising: i) inserting a seedling within the microfluidic device of the invention under conditions adequate for a root to develop within the substrate sheet in a direction which is substantially perpendicular to the direction of the channels within the device, ii) allowing the root to develop until it reaches in length the position of at least one of the channels in the device and iii) collecting and / or analyzing the exudates from one or more of said channels, simultaneously, after a predefined period of root development.

[0015] In another aspect, the present invention relates to the use of the microfluidic device of the invention for spatial and / or temporal detection of root exudates and / or root microbiome exudates.

[0016] In another aspect, the present invention relates to a method for the manufacture of the microfluidic device of the invention comprising: i) contacting the slide of the elastomeric material with the side of the substrate sheet which is the opposite of the side that contains the deposits of hydrophobic material that defined the patterned channels and wherein the slide of elastomeric material has been activated so that it can bind to the backing slide, ii) contacting the slide of elastomeric material to which the substrate sheet is adhered to with a backing slide, wherein the backing slide has been activated so that it binds to the slide of elastomeric material at those regions that are not covered by the substrate sheet; wherein the contacting step (ii) is carried out in order to provide sufficient contact for the formation of bonds between the slide of elastomeric material which is not covered with the substrate sheet and the backing slide.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. Working principle and assembly of wax printed paper-based microfluidic chip. A) Filter paper is laser cut to produce inlet (right of the chip) and outlet (left of the chip) of water. Wax patterns are printed on the paper to direct the flow of water that is created by evaporation at the outlet. The paper is placed into a chamber (consists of a glass and a PDMS slide) so that plant roots can grow healthily for a few days in contact with the paper. B) A sensor is placed on the paper at the flow outlet and image analysis allows automatic quantification of the state of the sensor C). D) Growing roots deposit biomolecules on the paper. These move with water to the left part of the paper. The wax patterns separate the different areas from the root and transport the biomolecules towards the sensors, from right to left.

[0019] Figure 2. Characterisation of the flow in the chip. A) Food dyes were used to test the speed of the flow in the channels. B) Flow speed as a function of time. Shaded areas correspond to the standard deviation of the flow speed (n = 3) for each dye. Yellow dye represented in light grey and Blue dye represended in darck grey.

[0020] Figure 3. Testing and calibration of measurements of glucose concentration. A) Signals were recorded from solution of known concentration of glucose for calibration. The darker colour indicates higher glucose concentrations. B) A calibration curve was established using the Hill-equation fitted on image saturation data. Error bars indicate the standard deviation (n = 5). C) Signals produced by the sensor as a function of time indicated the time needed for all available glucose accumulated in the paper to reach the sensor. D) Position of the sample during root exudate extraction. Figure 4. Glucose exudation patterns in two wheat varieties as a function of time. A) Glucose exudation of rgt Tocayo variety. B) Glucose exudation of Filon variety. C) Control experiment.

[0021] Figure 5. Glucose concentration in root exudates as a function of time and position along the root. A) Glucose concentration values for the different channels (Ch) over time for the rgt Tocayo variety. B) Glucose concentration values for the different channels over time for the Filon variety. The value of the control measurements is indicated with C. The error bars show the standard deviation (n=20). C) Cumulated exudation of rgt Tocayo variety as a function of time for the first and sixth channels. The grey and the black shadows correspond to the standard deviation of the saturation; n = 20 and n = 17, respectively. D) Cumulated exudation of Filon variety as a function of time for the first and sixth channels. The grey and the black shadows correspond to the standard deviation of the saturation; n = 12 and n = 16, respectively.

[0022] Figure 6. A) First, the PDMS mixture was introduced into a mould to get 2 mm thick PDMS sheets. B) One mould served for four PDMS slides, and the slices were cut using a scalpel. C) The PDMS and glass slides after activation. Then, the paper channels are placed onto the activated glass slide. D) Assembly of the microfluidic chip.

[0023] Figure 1. Preparation of the sensors in the paper surface, A) Scheme of the preparation of the glucose sensors. The TNT-Alginate hydrogel was pipetted onto the paper surface. 2 pL into each of the wax circles. B) For the jellification of the alginate, the sensor stripes were immersed into the CaCh solutions for 1 min and rinsed in water for another minute. C) Picture of a set of glucose sensors in a circular wax printed configuration. The dark colour corresponds to sensors that have detected glucose.

[0024] Figure 8. Design of the proposed sampling paper sheet for the paper microfluidic device.

[0025] Figure 9. The working principle of the sampling paper. The paper hexagonals inserted to the outlets. The dotted line shows the extended size of the PDMS.

[0026] Figure 10. (1) Scheme of the mechanism of action of the sampler. A) device where the exudate moves from de inlet to the outlet, collection zone; (B) sampler is manually positioned at the outlet. (C) exudate is transferred by capillary forces from the device to the sampler (D) exudate is accumulated in the hexagonal parts of the sampler. (E) sampler is removed and send for analisys. This determines the time: 1. The process can be repeated n times to monitor the exudate over time. (2) The saturation image of the accumulated food dye. The original image was converted to HSB image and the saturation values are presented. The brighter areas show the accumulated food dye after 24h at the top side of the hexagon.

[0027] Figure 11. Content of root exudates extracted from the different channels of the developed microfluidic device, ranges 0-20000 ph L'1and its zoom plot for the range 0- 1000 ph L’1.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The authors of the present invention have developed a microfluidic device designed to collect and / or analyse root exudates from precise location along the root.

[0030] Microfluidic device

[0031] In a first aspect the present invention relates to a microfluidic device, hereinafter the microfluidic device of the invention, comprising: i) a slide of an elastomeric material ; ii) a substrate sheet which contains a pattern of substantially parallel channels, wherein the channels in the pattern are defined by the space between regions in which a hydrophobic material has been deposited onto the substrate; and iii) a backing slide; wherein the substrate sheet is placed between the slide of elastomeric material and the backing slide, wherein the slide of elastomeric material and / or the backing slide is transparent, and wherein the backing slide and the slide of the elastomeric material are bonded to each other at those regions in which the substrate sheet does not separate the two surfaces thereby creating an inlet and an outlet at the opposite ends of each channel in the device.

[0032] The term “microfluidics” refers to designing, manufacturing and formulating devices and processes that deal with volumes of fluids, normally on the order of nanoliters or microliters. A “microfluidic device”, as used herein, refers to any device or system that allows for the precise control and manipulation of fluids that are geometrically constrained to structures in which at least one dimension (width, length, height) is less than 1 mm.

[0033] The microfluidic device of the invention comprises i) a slide of an elastomeric material.

[0034] The term "elastomeric" or "flexible," when applied to polymers, refers to a polymer with a modulus of elasticity, or Young's Modulus, or flexural modulus, below 0.1 GPa, i.e. a material being able to regain its original shape when a load is removed from the material. It is related to having the properties of elastomers. An “elastomer” is a polymer with viscoelasticity (i.e. both viscosity and elasticity) and with weak intermolecular forces. Elastomers are amorphous polymers maintained above their glass transition temperature, so that considerable molecular reconformation is feasible without breaking of covalent bonds. Rubber-like solids with elastic properties are called elastomers. Polymer chains are held together in these materials by relatively weak intermolecular bonds, which permit the polymers to stretch in response to macroscopic stresses. Materials possessing elastomeric properties include both natural and synthetic rubber, urethane, polyurea and polymeric coatings.

[0035] The choice of materials typically depends upon the particular material properties (e.g., solvent resistance, stiffness, gas permeability, and / or temperature stability) required for the application being conducted. In some embodiments, elastomeric materials that are used in the manufacture of components of the microfluidic devices are described in Unger (2000) Science 288:113-116, incorporated herein by reference in its entirety. Some elastomers of the present devices are used as diaphragms. In some embodiments, elastomers are selected for their porosity, impermeability, chemical resistance, wetting, and passivating characteristics in addition to their stretch and relax properties.

[0036] Elastomeric polymers for use according to the present invention include, polyisoprene, polybutadiene, polychlorophene, polyisobutylene, poly(styrene-butadiene-styrene), nitriles, polyurethanes, or polysilicones.

[0037] In a particular embodiment, the elastomeric material is a silicon-based polymer, such as PDMS. PDMS, also known as dimethylpolysiloxane or dimethicone, is the linear polymer of dimethylsiloxane. It belongs to the group of organosilicon compounds, substances commonly known as silicones. PDMS is optically clear and, in general, inert, non-toxic, and non-flammable.

[0038] In another particular embodiment, the elastomeric material is an amorphous polymer.

[0039] The term “amorphous polymers” refers to polymers that are composed of amorphous regions where molecules are randomly arranged. Polymers can be either completely amorphous or mixed with both amorphous and crystalline regions. Amorphous polymers possess widely different mechanical and physical properties owing to their structure and temperature. In a particular embodiment, the amorphous polymer is a cyclic olefin copolymer (COC) or cyclo olefin polymer (COP).

[0040] In another particular embodiment, the elastomeric material is a thermoplastic, such as poly(methyl methacrylate) (PMMA), polyethylene (PE) or polyether ether ketone (PEEK). The term “thermoplastic”, or “thermosoft plastic”, is any plastic polymer material that becomes pliable or moldable at a certain elevated temperature and solidifies upon cooling. Most thermoplastics have a high molecular weight. The polymer chains associate by intermolecular forces, which weaken rapidly with increased temperature, yielding a viscous liquid. In a particular embodiment, the thermoplastic is selected from the group consisting of: PMMA, PE, acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polybenzimidazole (PBI), polycarbonate (PC), polyether sulfone (PES), polyoxymethylene (POM), polyetherimide (PEI), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polypropylene (PP), polyvinyl chloride (PVC), polyvinylidene fluoride( PVDF) or PEEK.

[0041] In a preferred embodiment, the elastomeric material is polydimethylsiloxane (PDMS). Thus, in a preferred embodiment, the slide of elastomeric materal in the microfluidic device of the invention is a PDMS slide.

[0042] In a more preferred embodiment, the slide of the elastomeric material is a 26 mm x 76 mm x 2 mm slide of PDMS.

[0043] In a particular embodiment, the slide of elastomeric material is transparent.

[0044] The microfluidic device of the invention comprises ii) a substrate sheet which contains a pattern of substantially parallel channels.

[0045] A variety of substrates can be useful in microfluidic devices. The term “substrate,” as used herein, refers to a material that forms the structural components of a microfluidic device and in which the microfluidic channels, microfluidic chambers, and / or the microwells are formed.

[0046] Examples of suitable substrates include cellulose; derivatives of cellulose such as nitrocellulose or cellulose acetate; paper (e.g., craft paper, card stock, filter paper, chromatography paper); woven cellulosic materials; non-woven cellulosic materials; and thin films of wood. In a preferred embodiment of the present invention, the substrate is a cellulosic material and, more preferably, paper.

[0047] Other examples of suitable substrates include textiles suchs as wool, silk, cotton, polyester, nylon, acrylic, rayon, viscose, bamboo, among others.

[0048] The term “paper” as used herein refers to a web of cellulosic fibers that are formed, for example, from an aqueous suspension on a wire or screen, and are held together at least in part by hydrogen bonding. Papers can be manufactured by hand or by machine. Paper can be formed from a wide range of matted or felted webs of vegetable fiber, such as “tree paper” manufactured from wood pulp derived from trees, as well as “plant papers” or “vegetable papers” which include a wide variety of plant fibers (also known as “secondary fibers”), such as straw, bamboo, flax, and rice fibers. Paper can be formed from substantially all virgin pulp fibers, substantially ail recycled pulp fibers, or both virgin and recycled pulp fibers. Paper can also materials generally referred to as “nonwovens”. Paper can also include adhesives, fillers, dyes, and other additives.

[0049] A variety of grades of paper are available, permitting the selection of a paper substrate with the weight (i.e., grammage), thickness and / or rigidity and surface characteristics, desired for the fabrication of a particular microfluidic device. Suitable papers can include, but are not limited to, chromatography paper, card stock, filter paper, vellum paper, printing paper, wrapping paper, ledger paper, bank paper, bond paper, blotting paper, drawing paper, fish paper, tissue paper, paper towel, wax paper, and photography paper.

[0050] The term “channel” refers to any means through which a stream can be flowed, preferably without leakage of said stream, such as tubes, pipes or capillaries. The channels are comprised in a microfluidic device, hence it is a microfluidic channel. Microfluidic channels can have at least one cross-sectional dimension that is in the range from about 0.1 pm to about 1000 pm. In some embodiments, microfluidic devices comprise channels having a channel depth of approximately 10-500 pm (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 pm). In some embodiments, microfluidic devices comprise channels comprising a channel width of approximately IQ- 500 pm (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 spm). The width of the channels in the microfluidic device of the invention can be adjusted according to the use of the microfluidic device. Microfluidic channels can include open channels, closed channels, or both open and closed channels. Microfluidic channels can also be made of porous paper or other material transmitting directionally the fluid.

[0051] The number of channels in the microfluidic device of the invention can be set as required. In a particular embodiment, the microfluidic device of the invention comprises between 2 and 50 channels, preferably between 2 and 10 channels, more preferably 6 channels. The channels may be arranged on the microfluidic device in parallel and, preferably, at equal intervals. In an embodiment, the microfluidic device has channels of different lengths. In another embodiment, the channels have the same length.

[0052] As an expert may know, different channels geometry are possible. In some embodiments, the channels are straight channels, In other embodiments, the channels are oblique channels. In other embodiments, the channels are curved channels.

[0053] In a particular embodiment, the width of the channels are between 250 pm and 1 cm, preferably between 1mm and 1 cm, more preferably between 5 mm and 1 cm. In a more particular embodiment, the channels are 8 mm wide.

[0054] As mentioned above, the channels contained in the pattern are defined by the space between regions in which a hydrophobic material has been deposited onto the substrate, preferably paper.

[0055] The term “hydrophobic” refers to the property of having a lack of affinity for, or even repelling water. As a result, hydrophobic surfaces have a tendency not to be wetted by water. In certain embodiments, hydrophobic surfaces or substrates have a water contact angle of greater than 90°.

[0056] The term “hydrophobic material” as used herein refers to non-polar materials with a low affinity to water, which makes them water repelling. In a particular embodiment, the hydrophobic material is selected from the group consisting of: hydrophobic inks, such as CytoPel, CytoCure, CytoSyl, Staedtler Lumocolor and PVC ink, hydrophobic polymers such as acrylics, epoxides, acrylics, polyethylene, polystyrene, polyvinylchloride, polytetrafluoroethylene, polydimethylsiloxane, polyesters and polyurethanes, and wax.

[0057] In a more particular embodiment, the hydrophobic material is wax.

[0058] The term “waxes” or “wax” refers to a diverse class of organic compounds that are lipophilic, malleable solids near ambient temperatures. They include higher alkanes and lipids, typically with melting points above about 40 °C (104 °F), melting to give low viscosity liquids. Waxes are insoluble in water but soluble in nonpolar organic solvents such as hexane, benzene and chloroform. Waxes are organic compounds that characteristically consist of long aliphatic alkyl chains, although aromatic compounds may also be present.

[0059] In a particular embodiment, the wax is a natural wax. Natural waxes may contain unsaturated bonds and include various functional groups such as fatty acids, primary and secondary alcohols, ketones, aldehydes and fatty acid esters. In a more particular embodiment, the natural wax is an animal wax, a plant wax or a modified plant or animal wax. Plant and animal based waxes or oils can undergo selective chemical modifications to produce waxes with more desirable properties than are available in the unmodified starting materia.

[0060] In another particular embodimnet, the wax is a petroleum derived wax or a paraffin wax. Paraffin waxes are hydrocarbons, mixtures of alkanes usually in a homologous series of chain lengths. Paraffin waxes are mixtures of saturated n- and iso- alkanes, naphthenes, and alkyl- and naphthene-substituted aromatic compounds.

[0061] In another particular embodiment, the wax is a synthetic wax. Synthetic waxes often consist of homologous series of long-chain aliphatic hydrocarbons (alkanes or paraffins) that lack functional groups.

[0062] In the microfluidic device of the invention, the width of the deposits of the hydrophobic mateial, preferably wax, can be adjusted. In the device, the hydrophobic barrier, preferably wax barrier, has to be strong enough to separate the channels from each other. Thus, in a particular embodiment, the width of the deposits of the hydrophobic material is of at least 1 ,5 mm, preferably 2 mm.

[0063] In a particular embodiment, the microfluidic device of the invention comprises a substrate sheet, preferably a sheet of filter paper, which contains a pattern of parallel channels of between 250 pm and 1 cm wide, preferably between 1 mm and 1 cm wide, more preferably between 5 mm and 1 cm wide, even more preferably 8 mm wide, wherein the width of the deposits of the hydrophobic material, in particular wax, is of at least 1 ,5 mm, preferably 2 mm.

[0064] In a more particular embodiment, the microfluidic device of the invention comprises a substrate sheet, preferably a sheet of filter paper, which contains a pattern of parallel channels of 8 mm wide, wherein the filter paper has deposits of the hydrophobic material, preferably wax, of 2 mm wide.

[0065] The microfluidic device of the invention comprises iii) a backing slide. In a particular embodiment, the backing slide is transparent.

[0066] A material is “transparent” when it allows light to pass through easily. In a particular embodiment, the transparent material is selected from the group consisting of: glass, pressure-sensitive adhesive (PSA), quartz, foils, PMMA, PEEK, PLA, PC or PE. In a more particular embodiment, the transparent backing slide is a glass slide. In the microfluidic device of the invention the substrate sheet is placed between the slide of elastomeric material and the backing slide. Thus, in a particular embodiment, the substrate sheet is placed between the PDMS slide and the glass slide.

[0067] In the microfluidic device of the invention, the backing slide and the slide of the elastomeric material are bonded to each other at those regions in which the substrate sheet does not separate the two surfaces thereby creating an inlet and an outlet at the opposite ends of each channel in the device. Thus, in a particular embodiment, the glass slide and the PDMS slide are bonded to each other at those regions in which the paper does not separate the two surfaces.

[0068] An “inlet” or an “outlet”, as used herein, may include any or an aperture whereby fluid flow is restricted through the inlet, outlet or aperture. The inlets and outlets are configured for receiving a liquid sample, such as water or a buffer. The solution may be injected inside the microfluidic device through the inlet of the device.

[0069] In some embodiments, the inlets can be adapted to receive a liquid sample deposited from a pipette.

[0070] In a particular embodiment, the inlets are between 3 mm and 8 mm wide, preferably 4 mm wide, and between 8 mm and 15 mm long, preferably 10,4 mm long.

[0071] In another particular embodiment, the outlets are between 3 mm and 8 mm wide, preferably 4 mm wide, and between 9 mm and 18 mm long, preferably 15,8 mm long.

[0072] The microfluidic device of the invention has as many outlets as microchannels so that one and only microchannel connects the inlet chamber to one and only corresponding outlet chamber.

[0073] In a particular embodiment, the number of outlets and the number of channels is between 2 and 50, preferably between 6 and 10, more preferably 6.

[0074] The microfluidic device can include one or more sensors and / or one or more assay regions. Thus, in a particular embodiment, the microfluidic device of the invention further comprises one or more sensors which are connected to each of the outlets of the device.

[0075] The term “sensor” as used herein refers to a device that produces an output signal for the purpose of sensing a physical phenomenon.

[0076] Assay regions can include one or more assay reagents that serve as indicators for the presence of one or more analytes. The one or more assay regions may also include a sensor or electrode assembly that can be used to detect or quantify one or more analytes within a liquid sample. Sensors can include suitable optical sensors, electrochemical sensors, fluorescent sensors, impedance sensors, turbidimetric sensors, or a combination thereof. The sensors can include an electrode assembly. In these embodiments, the one or more electrodes can be fabricated from suitable conductive materials, including carbon ink, silver ink, Ag / AgCI ink, copper, nickel, tin, gold, platinum, and combinations thereof.

[0077] Suitable assay reagents can be included in the microfluidic device to be used in conjunction with the sensors and / or electrode assemblies. Assay reagents can serve as indicators for the presence of one or more analytes. In certain embodiments, the assay reagents facilitate the detection and / or quantification of one or more analytes, such as small molecules, proteins, lipids, polysaccharides, nucleic acids, prokaryotic cells, eukaryotic cells, particles, viruses, fungi, metal ions, or combinations thereof.

[0078] In certain cases, the microfluidic devices can be intended to detect and / or quantify one or more analytes without the use of complicated and expensive instrumentation. In these instances, the one or more assay reagents can be selected so as to provide a response that is visible to the naked eye. For example, the assay reagent can be an indicator that exhibits colorimetric and / or fluorometric response in the presence of the analyte of interest. Indicators can include molecules that become colored in the presence of the analyte, change color in the presence of the analyte, or emit fluorescence, phosphorescence, or luminescence in the presence of the analyte. In some embodiments, the one or more assay reagents are selected to facilitate radiological, magnetic, optical, and / or electrical measurements used to identify and / or quantify one or more analytes in a liquid sample.

[0079] In a particular embodiment, the sensor is selected from the group consisting of: a colorimetric sensor, such as a glucose sensor or a pH sensor, a fluorescent sensor and an electrochemical sensor.

[0080] Colorimetric sensors are a class of optical sensors that change their color when influenced by external stimuli. Any change in a physical or chemical environment can be considered as such stimulus. In a particular embodiment, the colorimetric sensor is a glucose sensor or a pH sensor. In a more particular embodiment, the sensor is a glucose sensor.

[0081] In a still more particular embodiment, the glucose sensor is a colorimetric glucose sensor, preferably an alginate / TiCh hydrogel based glucose sensor.

[0082] The alginate / TiC>2 hydrogel based glucose sensor is a colorimetric sensor, its initial colour is white and the presence of glucose changes its colour to blue, with the colour intensity relating to glucose concentration. In this sensor, the enzyme complex was encapsulated in a sodium-alginate hydrogel matrix with additional titanate nanotubes (TNT). The enzyme complex used in the alginate / TiCh hydrogel based glucose sensor comprises glucose oxidase (horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB).

[0083] In a particular embodiment, the outlets of the channels are connected to collecting means which allow the collection of the fluid which exists through the outlets.

[0084] A variaty of collecting means can be used. The collecting means can be made from cellulose; derivatives of cellulose such as nitrocellulose or cellulose acetate; paper (e.g., craft paper, card stock, filter paper, chromatography paper); woven cellulosic materials; non-woven cellulosic materials; and thin films of wood. In a particular embodiment, the collecting means are made of paper.

[0085] The collecting means can have different shapes. In a particular embodiment, the collecting means have a polygonal shape. “Polygonal shapes” include but are not limited to, triangular shape, quadrilateral shape, circular shape, pentagonal shape, hexagonal shape, heptagonal shape, octagonal shape or other.

[0086] In a particular embodiment, the collecting means have hexagonal shape. In a more particular embodiment, every side of the hexagons are between 3 mm and 4 mm long, preferably 3.6 mm long.

[0087] In a particular embodiment, the collecting means are connected to the channel outlets by paper connectors. In a more particular embodiment, the collecting means are inserted to the paper outlet channels through short paper bridges.

[0088] Method for analysing or collecting root exudates

[0089] In another aspect, the present invention relates to a method, hereinafter the first method of the invention, for analysing or collecting root exudates and / or root microbiome exudates from different regions within the longitudinal axis of a root, the method comprising: i) inserting a seedling within the microfluidic device of the invention under conditions adequate for a root to develop within the substrate sheet in a direction which is substantially perpendicular to the direction of the channels within the device, ii) allowing the root to develop until it reaches in length the position of at least one of the channels in the device, and iii) collecting and / or analyzing the exudates from one or more of said channels, simultaneously, after a predefined period of root development.

[0090] The term “root” refers to the part of a plant that is usually buried in the soil. Roots are usually always under the soil, though sometimes roots can be above the ground. This is called an aerial root.

[0091] The term “root exudates” refers to fluids emitted through the roots of plants. These secretions influence the rhizosphere around the roots to inhibit harmful microbes and promote the growth of self and kin plants.

[0092] Root exudates are a mix of a wide variety of compounds, including nucleic acids, RNA, DNA molecules, proteins, carbohydrates, saccharides, polysaccharides, fatty acids, hormones, amino acids, organic acids, flavonoids, glucosinolates, auxins, etc.

[0093] The term “root exudates” as used in the present invention includes, but is not limited to, amino acids such as a-Alanine, p-alanine, y-aminobutyric, a-aminoadipic, arginine, asparagine, aspartic, citrulline, cystathionine, cysteine, cystine, deoxymugineic, 3- epihydroxymugineic, glutamine, glutamic, glycine, histidine, homoserine, isoleucine, leucine, lysine, methionine, mugineic, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; sugars such as arabinose, fructose, galactose, glucose, maltose, mannose, mucilages of various compositions, oligosaccharides, rafnose, rhamnose, ribose, sucrose, xylose and deoxyribose; organic acids such as acetic, aconitic, ascorbic, aldonic, benzoic, butyric, cafeic, citric, p- coumaric, erythronic, ferulic, formic, fumaric, glutaric, glycolic, lactic, glyoxilic, malic, malonic, oxalacetic, oxalic, p-hydroxybenzoic, piscidic, propionic, pyruvic, succinic, syringic, tartaric, tetronic, valeric and vanillic; fatty acids, such as linoleic, linolenic, oleic, palmitic and stearic; sterols such as campesterol, cholesterol, sitosterol and stigmasterol; growth factors and vitamins such as p-amino benzoic acid, biotin, choline, inositol, N-methyl nicotinic acid, niacin, pathothenic, pantothenate, pyridoxine ribofavin, strigolactones and thiamine; enzymes such as amylase, invertase, peroxidase, phenolase, acid / alkaline phosphatase, polygalacturonase and protease; flavonoids such as chaicone, coumarine, favones, favonols, favanones, favonones and isofavones; nucleotides such as adenine, guanine and uridine / cytidine; Al-induced polypeptides, alcohols, alkyl sulphides, auxins, camalexin, dihydroquinone, ethanol, glucosides, glucosinolates, glycinebetaine, hydrocyanic acid, inorganic ions and gaseous molecules (e.g. CO2, H2, H+, OH-, HCO3), isothiocyanates, unidentifed ninhydrin positive compounds, unidentifable soluble proteins, reducing compounds, scopoletin, sorgoleone and strigolactones.

[0094] The “rhizosphere” is the thin area of soil immediately surrounding the root system. It is a densely populated area in which the roots compete with invading root systems of neighboring plant species for space, water, and mineral nutrients as well as form positive and negative relationships with soil-borne microorganisms such as bacteria, fungi and insects.

[0095] In the rhizosphere and the soil, roots are not the sole actors to release exudates. The microorganisms, bacteria, fungi or archaea, and even insects release different chemicals and all of them can originate exudates which influence the life of the plant too. The microfluidic device of the invention has the capability to extract these exudates as well. The working principle is the same. They release their exudates into the environment, particularly into the paper tissue, and the exudates can be extracted from the channels together with the plant root exudates.

[0096] The term “root microbiome exudates” refers to the secretion products of microorganisms and / or instects associated with the root. Examples of root microbiome exudates are sugars, pigments, amino acids, organic acids, nucleic acids or chemical compounds, such as nitric oxide.

[0097] The “root microbiome” or “rhizosphere microbiome” is the dynamic community of microorganisms and / or insects associated with plant roots. Because they are rich in a variety of carbon compounds, plant roots provide unique environments for a diverse assemblage of soil microorganisms, including bacteria, fungi and archaea. In addition, other organisms such as viruses, algae, protozoa, nematodes and arthropods are part of root microbiota.

[0098] In a particular embodiment, the root microbiome exudates are produced by bacteria, fungi, archaea, viruses, algae, protozoa, nematodes, arthropods or a combination thereof. In a more particular embodiment, the root microbiome exudates are produced by bacteria, fungi, archaea or a combinarion thereof.

[0099] Fungi that extend beyond the root surface and engage in nutrient-carbon exchange with the plant host are commonly considered to be mycorrhizal, but external hyphae can also include other endophytic fungi. Mycorrhizae include a broad variety of root-fungi interactions characterized by mode of colonization. Essentially all plants form mycorrhizal associations, and there is evidence that some mycorrhizae transport carbon and other nutrients not just from soil to plant, but also between different plants in a landscape. The main groups include ectomycorrhizae, arbuscular mycorhizae, ericoid mycorrhizae, orchid mycorrhizae, and monotropoid mycorrhizae.

[0100] Endophytes grow inside the plant tissues — roots, stems, leaves — mostly symptomless; however, when plant ages they can become slightly pathogenic. They may colonize intercellular spaces, the root cells themselves, or both. Rhizobia and dark septate endophytes (which produce melanin, an antioxidant that may provide resilience against a variety of environmental stresses) are some examples.

[0101] In a particular embodiment, the root microbiome exudates are produced by fungi, preferably mycorrhizal and / or an endophyte fungi.

[0102] In a more particular embodiment the root microbiome exudates are produced by a fungi selected from: an ectomycorrhizae, an arbuscular mycorhizae, an ericoid mycorrhizae, an orchid mycorrhizae, a monotropoid mycorrhizae and / or an endophyte fungi, such as endophytes in the phyla Basidiomycota and Ascomycota, Sordariomycetes and Loculoascomycetes class, Glomeromycota division.

[0103] Root associated beneficial bacteria promote plant growth and provide protection from pathogens. They are mostly rhizobacteria that belong to Pseudomonadota and Bacillota, with many examples from Pseudomonas and Bacillus genera. Rhizobium species colonize legume roots forming nodule structures. In response to root exudates, rhizobia produce Nod signalling factors that are recognized by legumes and induce formation of nodules on plant roots. Within these structures Rhizobium perform nitrogen fixation that proves plant with nitrogen source. In turn, plants provide bacteria with carbon source to energize the nitrogen fixation. In addition to nitrogen fixation, Azospirillum species promote plant growth through production of growth phytohormones (auxins, cytokinins, gibberellins). Due to these phytohormones root hairs expand to occupy larger area and better acquire water and nutrients.

[0104] In a particular embodiment, the root microbiome exudates are produced by bacteria, preferably bacteria from the genus Pseudomonas, Bacillus, Rhizobium, Azospirillum, Agro bacterium, Pecto bacterium and / or Ralstonia.

[0105] Though archaea are traditionally thought of as extremophiles, microbes belonging to extreme environments, advances in metagenomics and gene sequencing reveal that archaea are ubiquitous, found in nearly any environment including the root microbiome. Methanogen and ammonium-oxidizing archaea are prevalent members of the root microbiome, especially in anaerobic soils and wetlands. Archaeal phyla found in the root microbiome include Euryarchaeota, Nitrososphaerota and Thermoproteota. In a particular embodiment, the root microbiome exudates are produced by a archaea, preferably from the phyla Euryarchaeota, Nitrososphaerota or Thermoproteota.

[0106] In another particular embodiment, the root microbiome exudates are produced by an ectomycorrhizae, an arbuscular mycorhizae, an ericoid mycorrhizae, an orchid mycorrhizae, a monotropoid mycorrhizae and / or an endophyte fungi (such as endophytes in the phyla Basidiomycota and Ascomycota, Sordariomycetes and Loculoascomycetes class, Glomeromycota division), by a bacteria from the genus Pseudomonas, Bacillus, Rhizobium, Azospirillum, Agrobacterium, Pecto bacterium and / or Ralstonia, and / or by a archaea, preferably from the phyla Euryarchaeota, Nitrososphaerota or Thermoproteota.

[0107] The first step of the first method of the invention comprises inserting a seedling within the microfluidic device of the invention under conditions adequate for a root to develop within the substrate sheet in a direction which is substantially perpendicular to the direction of the channels within the microfluidic device.

[0108] The term “seedling” refers to a a young sporophyte developing out of a plant embryo from a seed. Seedling development starts with germination of the seed. The term “germination” refers to the sprouting of a seed after a period of dormancy. Seed germination depends on both internal and external conditions. The most important external factors include right temperature, water, oxygen or air and sometimes light or darkness. Various plants require different variables for successful seed germination. Often this depends on the individual seed variety and is closely linked to the ecological conditions of a plant's natural habitat.

[0109] In a particular embodiment, the seedling is inserted in the microfluidic device of the invention at least after one day of germination. In some embodiments, the seedling is inserted in the microfluidic device of the invention after one day of germination, after 2 days of germination, after 3 days of germination, after 4 days of germination, after 5 days of germination, after 6 days of germination, after 7 days of germination, after 8 days of germination, after 9 days of germination or after 10 days of germination. In a preferred embodiment, the seedling is inserted in the microfluidic device of the invention after one day of germination.

[0110] The seedling that is inserted in the microfluidic device can be selected from any plant. In a particular embodiment, the seedling is from a plant family selected from the group consisting of Solanaceae, Cucurbitaceae, Brassicacea, Poacea and Fabaceae. In a more particular embodiment, the seedling is from a plant genus selected from the group consisting of Arabidopsis, Solanum, Lycium, Citrullus, Cucumis, Cucurbita, Lactuca, Brassica, Spinacia, Oryza, Zea, Triticum, Sorghum, Secale, Hordeum, Saccharum, Lens, Cicer, Pisum, Prunus, Pistacia, Citrus, Musa, Vaccinium, Rubus and Olea.

[0111] In a still more particular embodiment, the seedling is selected from the species Arabidopsis thaliana, S. aviculare, S. capsicastrum, S. crispum, S. laciniatum, S. laxum, S. mammosum, S. pseudocapsicum, S. rantonnetii, S. seaforthianum, S. mauritianum, S. wendlandii, Lycium afrum, Lycium andersonii, Lycium australe, Lycium barbarum, Lycium berlandieri, Lycium brevipes, Lycium californicum, Lycium carolinianum, Lycium chinense, Lycium cinereum, Lycium cooperi, Lycium europaeum, Lycium horridum, Lycium intricatum, Lycium oxycarpum, Lycium pallidum, Citrullus amarus, Citrullus colocynthis, Citrullus ecirrhosus, Citrullus lanatus, Citrullus lanatus subsp. vulgaris var. cordophanus, Citrullus lanatus var lanatus, Citrullus mucosospermus, Citrullus naudinianus, Citrullus rehmii, Cucumis althaeoides, Cucumis anguria, Cucumis argenteus, Cucumis dipsaceus, Cucumis ficifolius, Cucumis humifructus, Cucumis hystrix, Cucumis maderaspatanus, Cucumis metuliferus, Cucumis myriocarpus, Cucumis picrocarpus, Cucumis prophetarum, Cucumis umbellatus, Cucumis variabilis, Curcubita argyrosperma, Curcubita digitata, Curcubita ecuadorensis, Curcubita ficifolia, Curcubita foetidissima, Curcubita galeottii, Curcubita lundelliana, Curcubita maxima, Curcubita moschata, Curcubita pedatifolia, Curcubita radicans, Lactuca aculeata, Lactuca acuminata, Lactuca alpestris, Lactuca alpina, Lactuca altaica, Lactuca biennis, Lactuca calophylla, Lactuca canadensis, Lactuca cyprica, Lactuca deltoidea, Lactuca dissecta, Lactuca dolichophylla, Lactuca graminifolia, Lactuca ludoviciana, Lactuca macrophylla, Lactuca orientalis, Lactuca palmensis, Lactuca perennis, Lactuca quercina, Lactuca saligna, Lactuca sativa, Lactuca tatarica, Lactuca tenerrima, Lactuca tetrantha, Brassica carinata, Brassica juncea, Brassica napus, Brassica nigra, Brassica oleracea, Brassica pekinensis, Brassica rapa, Brassica tournefortii, Spinacia oleracea, Oryza australiensis, Oryza barthii, Oryza brachyantha, Oryza eichingeri, Oryza glaberrima, Oryza grandiglumis, Oryza latifolia, Oryza longistaminata, Oryza officinalis, Oryza punctata, Oryza rufipogon, Oryza sativa, T. aestivum, T. hybernum, T. turgidum, T. spelta, T. monococcum, Zea diploperennis, Zea luxurians, Zea mays, Zea nicaraguensis, Zea perennis, Sorghum arundinaceum, Sorghum bicolor, Sorghum x drummondii, Sorghum halepense, Sorghum leiocladum, Sorghum macrospermum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum timorense, Secale cereale, Secale montanum, Secale vavilovii, Hordeum arizonicum, Hordeum brachyantherum, Hordeum brevisubulatum, Hordeum bulbosum, Hordeum capense, Hordeum chilense, Hordeum comosum, Hordeum cordobense, Hordeum depressum, Hordeum distichon, Hordeum lechleri, Hordeum marinum, Hordeum murinum, Hordeum muticum, Hordeum parodii, Hordeum patagonicum, Hordeum pubiflorum, Hordeum pusilium, Hordeum vulgare, Saccharum officinarum, Saccharum robustum, Saccharum x sinense Roxb., Saccharum spontaneum, Lens nigricans, Lens ervoides, Lens lamottei, Lens odemensis, Lens tomotosus, Lens orientalis, Lens culinaris, Cicer arietinum, Cicer reticulatum, Pisum abyssinicum, Pisum sativum, Pistacia aethiopica, Pistacia atlantica, Pistacia chinensis, Pistacia cucphuongensis, Pistacia eurycarpa, Pistacia lentiscus L., Pistacia mexicana, Pistacia terebinthus, Pistacia vera L., Citrus assamensis, Citrus crenatifolia, Citrus japonica, Citrus mangshanensis, Citrus maxima, Citrus medica, Citrus reticulata, Citrus trifoliata, M. azizii, M. barioensis, M. bauensis, M. beccarii, M. boman, M. borneensis, M. bukensis, M. velutina, M. campestris, M. coccinea, M. exotica, M. fitzalanii, M. gracilis, M. hirta, M. insularimontana, M. jackeyi, M. johnsii, M. lawitiensis, M. monticola, M. muluensis, M. paracoccinea, M. peekelii, M. salaccensis, M. textilis, M. x troglodytarum L., M. tuberculata, M. violascens, M. viridis, M. voonii, Vaccinium arboreum, Vaccinium crassifolium, Vaccinium bracteatum, Vaccinium angustifolium, Vaccinium boreale, Vaccinium caesariense, Vaccinium corymbosum, Vaccinium darrowii, Vaccinium elliottii, Vaccinium formosum, Vaccinium fuscatum, Vaccinium hirsutum, Vaccinium myrsinites, Vaccinium myrtilloides, Vaccinium pallidum, Vaccinium macrocarpon, Vaccinium oxycoccos, Vaccinium microcarpum, Vaccinium erythrocarpum, Vaccinium japonicum, Rubus aboriginum, Rubus allegheniensis, Rubus arcticus, Rubus argutus, Rubus armeniacus, Rubus caesius, Rubus canadensis, Rubus chamaemorus, Rubus cockburnianus, Rubus coreanus, Rubus crataegifolius, Rubus deliciosus, Rubus domingensis, Rubus ellipticus, Rubus flagellaris, Rubus fraxinifolius, Rubus glaucus, Rubus hawaiensis, Rubus hayata, Rubus hispidus, Rubus idaeus, Rubus illecebrosus, Rubus laciniatus, Rubus leucodermis, Rubus moluccanus, Rubus nepalensis, Rubus nivalis, Rubus niveus, Rubus occidentalis, Rubus odoratus, Rubus parviflorus, Rubus pedatus, Rubus pensilvanicus, Rubus phoenicolasius, Rubus probus

[0112] Rubus pubescens, Rubus rosifolius, Rubus saxatilis, Rubus spectabilis, Rubus tricolor, Rubus trivialis, Olea borneensis, Olea brachiata, Olea capensis, Olea chimanimani, Olea europaea, Olea lancea, Olea paniculata, Olea rubrovenia, Olea woodiana, Olea yuennanensis, P. africana, P. apetala, P. armeniaca, P. avium, P. brigantina, P. buergeriana, P. campanulata, P. canescens, P. cerasifera, P. cerasoides, P. cerasus, P. ceylanica, P. cocomilia, P. cornuta, P. davidiana, P. darvasica, P. domestica, P. dulcis, P. fruticosa, P. glandulosa, P. grayana, P. incana, P. incisa, P. jacquemontii, P. japonica, P. laurocerasus, P. lusitanica, P. mahaleb, P. mandshurica, P. maximowiczi, P. mume, P. nipponica, P. padus, P. persica, P. pseudocerasus, P. prostrata , P. sargentii, P. scoparia, P. serrula, P. serrulata, P. sibirica, P. simonii, P. tomentosa, P. triloba, P. turneriana, P. ursina, P. alabamensis, P. americana, P. andersonii, P. angustifolia, P. brasiliensis, P. buxifolia, P. caroliniana, P. cortapico, P. emarginata, P. eremophila, P. fasciculata, P. gracilis, P. havardii, P. hortulana, P. huantensis, P. ilici folia, P. nigra, P. occidentalis, P. pensylvanica , P. pleuradenia, P. pumila, P. rigida, P. rivularis, P. serotina, P. subcordata, P. subcorymbosa, P. texana, P. umbellata, P. virginiana.

[0113] In a preferred embodiment, the seedling is from the genus Triticum.

[0114] The genus Triticum includes the wild and domesticated species usually thought of as wheat. In a more preferred embodiment, the seedling is from the species T. aestivum, T. hybernum, T. turgidum, T. spelta or T. monococcum. In a more preferred embodiment, the seedling is from the species T. aestivum. In a still more preferred embodiment, the seedling is from the varieties of winter wheat Triticum aestivum var. rgt Tocayo or var. Filon.

[0115] The conditions suitable for the development of the root within the substrate sheet, preferably a sheet of filter paper, are well known for the person skilled in the art and will depend on the plant specie.

[0116] The development of the root may be afected by the humidity conditions, temperature and light / dark periods. As an expert may know, the humidity conditions, temperature and light / dark periords will vary depending on the plant.

[0117] In a particular embodiment, plants are kept at a temperature between 20°C and 24 °C, preferably at 22 °C.

[0118] In another particular embodiment, plants are kept under between 12-16 hours light and 8-12 hours dark periods.

[0119] In a more particular embodiment, plants are kept at 22°C, under 14 hours light and 10 hours dark periods.

[0120] The analysis and / or collection of the root exudates and / or the root microbiome exudates can be carried out in only one channel or in more than one channel of the microfluidic device.

[0121] In the first method of the invention, the root exudates and / or the root microbiome exudates from different channels are simultaneously collected or analysed after a predefined period of root development. This allows a spatial analysis of the exudates. In a particular embodiment, the root exudates and / or the root microbiome exudates from the different channels are collected or analysed in different time intervals. This allows a temporal anaysis of the exudates. Thus, in a particular embodiment, step (iii) is repeated several times at defined time intervals.

[0122] In a particular embodiment, the analysis is carried out using a sensor. The term “sensor” has been defined or explained in the microfluidic device of the invention, and this definiton is applicable to the first method of the invention.

[0123] In a particular embodiment, the sensor is connected to each of the outlets within the device.

[0124] Sensors can include suitable optical sensors, electrochemical sensors, fluorescent sensors, impedance sensors, turbidimetric sensors, or a combination thereof.

[0125] In a particular embodiment, the sensor is selected from the group consisting of: a colorimetric sensor such as a glucose sensor or a pH sensor, a fluorescent sensor and a electrochemical sensor.

[0126] In a more particular embodiment, the sensor is a glucose sensor.

[0127] In a still more particular embodiment, the glucose sensor is a colorimetric glucose sensor, preferably is an alginate / TiCh hydrogel based glucose sensor. The alginate / TiCh hydrogel based glucose sensor has been defined or explained in the microfluidic device of the invention and this definition is applicable to the first method of the invention.

[0128] The second step of the first method of the invention comprises allowing the root to develop until it reaches in length the position of at least one of the channels in the microfluidic device.

[0129] In a particular embodiment, the root exudates and / or the root microbiome exudates are collected or analysed when the root reaches in length the position of at least one of the channels in the microfluidic device.

[0130] In a particular embodiment, the incubating step ii) is carried out for a period of time that allows the root of the seedling to reach the channel of the microfluidic device which is opposite to the region in the device wherein the seedling is inserted.

[0131] In a particular embodiment, step ii) is carried out for a period of between 7 days and 15 days.

[0132] Thus, in a particular embodiment, the root exudates and / or the root microbiome exudates from different channels are simultaneosly collected or analysed when the root reaches the channel of the microfluidic device which is opposite to the region in the device wherein the seedling is inserted.

[0133] In another particular embodiment, the root exudates and / or the root microbiome exudates from different channels are simultaneosly collected or analysed at least 7 days after the seedling is inserted in the microfluidic device.

[0134] In a more particular embodiment, the root exudates and / or the root microbiome exudates from different channels are simultaneosly collected or analysed between 7 days and 15 days after the seedling is inserted in the microfluidic device.

[0135] The third step of the first method of the invention comprises collecting and / or analyzing the exudates from one or more of said channels, simultaneously, after a predefined period of root development.

[0136] In some embodiments, after at least 7 days of incubation, the microfluidic device of the invention with the plants are placed into Petri-dishes. The inlets of the microfluidic device are immersed into the water, while the outlets left facing upward and outside of water. Water evaporation at the outlet created a gradient in water content, which resulted in a flow of water and exudates to the sensor by capillary action.

[0137] The term “capillarity” refers to the ability of a liquid to move through a small passage such as a tube of small cross-sectional area, like the spaces between the fibers of a towel or the openings in a porous materia.

[0138] Thus, in a particular embodiment, the channel outlets are open so that the exudates move towards the outlet as a result of evaporation of the liquid that pass through the channel open outlets from the inlet.

[0139] In a particular embodiment, sensors are connected to one or more outlets in the device. In some embodiments, sensors are connected to one outlet in the microfluidic device. In other embodiments, sensors are connected to at least 2 outlets in the microfluidic device. In other embodiment, sensors are connected to all outlets in the microfluidic device.

[0140] In a particular embodiment, the outlets of the channels are connected to collecting means which allow the collection of the exudates by capillarity.

[0141] A variaty of collecting means can be used. The collecting means can be made from cellulose; derivatives of cellulose such as nitrocellulose or cellulose acetate; paper (e.g., craft paper, card stock, filter paper, chromatography paper); woven cellulosic materials; non-woven cellulosic materials; and thin films of wood. In a particular embodiment, the collecting means are made of paper. The collecting means can have different shapes. In a particular embodiment, the collecting means have a polygonal shape. “Polygonal shapes” include but are not limited to, triangular shape, quadrilateral shape, circular shape, pentagonal shape, hexagonal shape, heptagonal shape, octagonal shape or other

[0142] In a particular embodiment, the collecting means have hexagonal shape. In a more particular embodiment, every side of the hexagons are between 3 mm and 4 mm long, preferably 3.6 mm long.

[0143] In a particular embodiment, the collecting means are connected to the channel outlets by paper connectors. In a more particular embodiment, the collecting means are inserted to the paper outlet channels through short paper bridges.

[0144] As an expert may know, after the extraction of the root exudates and / or root microbiome exudates, the paper pieces can be moved away from the outlets of the microfludiic device and the exudates can be dissolved from the collecting means for further analysis using conventional analytical techniques such as GC-MS or HPLC.

[0145] Uses of the microfluidic device of the invention

[0146] In another aspect, the present invention relates to the use of the microfluidic device of the invention for spatial and / or temporal detection of root exudates and / or root microbiome exudates.

[0147] The term “root exudates” and “root microbiome exudates” have been defined or explained in the first method of the invention and this definition is applicable to the use of the microfluidic device.

[0148] “Spatial detection” as used in the present invention refers to the detection of root exudates in different segments or regions within the longitudinal axis of a root.

[0149] “Temporal detection” as used in the present invention refers to the detection of root exudates in different time intervals.

[0150] In a particular embodiment, the root is from a plant family selected from the group consisting of Solanaceae, Cucurbitaceae, Brassicacea, Poacea and Fabaceae.

[0151] In a more particular embodiment, the root is from a plant genus selected from the group consisting of Arabidopsis, Solanum, Lycium, Citrullus, Cucumis Cucurbita, Lactuca, Brassica, Spinacia, Oryza, Zea, Triticum, Sorghum, Secale, Hordeum, Saccharum, Lens, Cicer, Pisum, Prunus, Pistacia, Citrus, Musa, Vaccinium, Rubus and Olea. In a still more particular embodiment, the root is selected from the plant species Arabidopsis thaliana, S. aviculare, S. capsicastrum, S. crispum, S. laciniatum, S. laxum, S. mammosum, S. pseudocapsicum, S. rantonnetii, S. seaforthianum, S. mauritianum, S. wendlandii, Lycium afrum, Lycium andersonii, Lycium australe, Lycium barbarum, Lycium berlandieri, Lycium brevipes, Lycium californicum, Lycium carolinianum, Lycium chinense, Lycium cinereum, Lycium cooperi, Lycium europaeum, Lycium horridum, Lycium intricatum, Lycium oxycarpum, Lycium pallidum, Citrullus amarus, Citrullus colocynthis, Citrullus ecirrhosus, Citrullus lanatus, Citrullus lanatus subsp. vulgaris var. cordophanus, Citrullus lanatus var lanatus, Citrullus mucosospermus, Citrullus naudinianus, Citrullus rehmii, Cucumis althaeoides, Cucumis anguria, Cucumis argenteus, Cucumis dipsaceus, Cucumis ficifolius, Cucumis humifructus, Cucumis hystrix, Cucumis maderaspatanus, Cucumis metuliferus, Cucumis myriocarpus, Cucumis picrocarpus, Cucumis prophetarum, Cucumis umbellatus, Cucumis variabilis, Curcubita argyrosperma, Curcubita digitata, Curcubita ecuadorensis, Curcubita ficifolia, Curcubita foetidissima, Curcubita galeottii, Curcubita lundelliana, Curcubita maxima, Curcubita moschata, Curcubita pedatifolia, Curcubita radicans, Lactuca aculeata, Lactuca acuminata, Lactuca alpestris, Lactuca alpina, Lactuca altaica, Lactuca biennis, Lactuca calophylla, Lactuca canadensis, Lactuca cyprica, Lactuca deltoidea, Lactuca dissecta, Lactuca dolichophylla, Lactuca graminifolia, Lactuca ludoviciana, Lactuca macrophylla, Lactuca orientalis, Lactuca palmensis, Lactuca perennis, Lactuca quercina, Lactuca saligna, Lactuca sativa, Lactuca tatarica, Lactuca tenerrima, Lactuca tetrantha, Brassica carinata, Brassica juncea, Brassica napus, Brassica nigra, Brassica oleracea, Brassica pekinensis, Brassica rapa, Brassica tournefortii, Spinacia oleracea, Oryza australiensis, Oryza barthii, Oryza brachyantha, Oryza eichingeri, Oryza glaberrima, Oryza grandiglumis, Oryza latifolia, Oryza longistaminata, Oryza officinalis, Oryza punctata, Oryza rufipogon, Oryza sativa, T. aestivum, T. hybernum, T. turgidum, T. spelta, T. monococcum, Zea diploperennis, Zea luxurians, Zea mays, Zea nicaraguensis, Zea perennis, Sorghum arundinaceum, Sorghum bicolor, Sorghum x drummondii, Sorghum halepense, Sorghum leiocladum, Sorghum macrospermum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum timorense, Secale cereale, Secale montanum, Secale vavilovii, Hordeum arizonicum, Hordeum brachyantherum, Hordeum brevisubulatum, Hordeum bulbosum, Hordeum capense, Hordeum chilense, Hordeum comosum, Hordeum cordobense, Hordeum depressum, Hordeum distichon, Hordeum lechleri, Hordeum marinum, Hordeum murinum, Hordeum muticum, Hordeum parodii, Hordeum patagonicum, Hordeum pubiflorum, Hordeum pusilium, Hordeum vulgare, Saccharum officinarum, Saccharum robustum, Saccharum x sinense Roxb., Saccharum spontaneum, Lens nigricans, Lens ervoides, Lens lamottei, Lens odemensis, Lens tomotosus, Lens orientalis, Lens culinaris, Cicer arietinum, Cicer reticulatum, Pisum abyssinicum, Pisum sativum, Pistacia aethiopica, Pistacia atlantica, Pistacia chinensis, Pistacia cucphuongensis, Pistacia eurycarpa, Pistacia lentiscus L., Pistacia mexicana, Pistacia terebinthus, Pistacia vera L., Citrus assamensis, Citrus crenatifolia, Citrus japonica, Citrus mangshanensis, Citrus maxima, Citrus medica, Citrus reticulata, Citrus trifoliata, M. azizii, M. barioensis, M. bauensis, M. beccarii, M. boman, M. borneensis, M. bukensis, M. velutina, M. campestris, M. coccinea, M. exotica, M. fitzalanii, M. gracilis, M. hirta, M. insularimontana, M. jackeyi, M. johnsii, M. lawitiensis, M. monticola, M. muluensis, M. paracoccinea, M. peekelii, M. salaccensis, M. textilis, M. x troglodytarum L., M. tuberculata, M. violascens, M. viridis, M. voonii, Vaccinium arboreum, Vaccinium crassifolium, Vaccinium bracteatum, Vaccinium angustifolium, Vaccinium boreale, Vaccinium caesariense, Vaccinium corymbosum, Vaccinium darrowii, Vaccinium elliottii, Vaccinium formosum, Vaccinium fuscatum, Vaccinium hirsutum, Vaccinium myrsinites, Vaccinium myrtilloides, Vaccinium pallidum, Vaccinium macrocarpon, Vaccinium oxycoccos, Vaccinium microcarpum, Vaccinium erythrocarpum, Vaccinium japonicum, Rubus aboriginum, Rubus allegheniensis, Rubus arcticus, Rubus argutus, Rubus armeniacus, Rubus caesius, Rubus canadensis, Rubus chamaemorus, Rubus cockburnianus, Rubus coreanus, Rubus crataegifolius, Rubus deliciosus, Rubus domingensis, Rubus ellipticus, Rubus flagellaris, Rubus fraxinifolius, Rubus glaucus, Rubus hawaiensis, Rubus hayata, Rubus hispidus, Rubus idaeus, Rubus illecebrosus, Rubus laciniatus, Rubus leucodermis, Rubus moluccanus, Rubus nepalensis, Rubus nivalis, Rubus niveus, Rubus occidentalis, Rubus odoratus, Rubus parviflorus, Rubus pedatus, Rubus pensilvanicus, Rubus phoenicolasius, Rubus probus

[0152] Rubus pubescens, Rubus rosifolius, Rubus saxatilis, Rubus spectabilis, Rubus tricolor, Rubus trivialis, Olea borneensis, Olea brachiata, Olea capensis, Olea chimanimani, Olea europaea, Olea lancea, Olea paniculata, Olea rubrovenia, Olea woodiana, Olea yuennanensis, P. africana, P. apetala, P. armeniaca, P. avium, P. brigantina, P. buergeriana, P. campanulata, P. canescens, P. cerasifera, P. cerasoides, P. cerasus, P. ceylanica, P. cocomilia, P. cornuta, P. davidiana, P. darvasica, P. domestica, P. dulcis, P. fruticosa, P. glandulosa, P. grayana, P. incana, P. incisa, P. jacquemontii, P. japonica, P. laurocerasus, P. lusitanica, P. mahaleb, P. mandshurica, P. maximowiczi, P. mume, P. nipponica, P. padus, P. persica, P. pseudocerasus, P. prostrata , P. sargentii, P. scoparia, P. serrula, P. serrulata, P. sibirica, P. simonii, P. tomentosa, P. triloba, P. turneriana, P. ursina, P. alabamensis, P. americana, P. andersonii, P. angustifolia, P. brasiliensis, P. buxifolia, P. caroliniana, P. cortapico, P. emarginata, P. eremophila, P. fasciculata, P. gracilis, P. havardii, P. hortulana, P. huantensis, P. ilici folia, P. nigra, P. occidentalis, P. pensylvanica , P. pleuradenia, P. pumila, P. rigida, P. rivularis, P. serotina, P. subcordata, P. subcorymbosa, P. texana, P. umbellata, P. virginiana.

[0153] In a preferred embodiment, the root is from the genus Triticum. In a more preferred embodiment, the root is from the species T. aestivum, T. hybernum, T. turgidum, T. spelta or T. monococcum. In a still more preferred embodiment, the root is from the species T. aestivum. In a still more preferred embodiment, the root is from the varieties of winter wheat Triticum aestivum var. rgt Tocayo or var. Pilon.

[0154] Method for the manufacture of the microfluidic device of the invention

[0155] The appropriate methods for fabrication of the microfluidic devices can be selected in view of the type of microfluidic features present in the device, as well as the overall device design.

[0156] Microfluidic devices can be fabricated into appropriate two- or three-dimensional shapes using a variety of methods. The substrates and / or covers can be mechanically cut, for example, by using a scissor, laser cutter, blade, knife, dye, or punch, to form a microfluidic device having the desired overall shape. In certain embodiments, the substrate and / or covers can also be perforated to facilitate folding or separation of the microfluidic devices after fabrication.

[0157] If desired, the shape of the device (and device components) can be designed on a computer using a layout editor or standard computer aided drafting software. The computer can be integrated with a laser cutter to automatically pattern the microfluidic device, and components thereof, into their desired shapes.

[0158] In another aspect, the present invention relates to a method, hereinafter the second method of the invention, for the manufacture of the microfluidic device of the invention comprising: i) contacting the slide of the elastomeric material with the side of substrate which is the opposite of the side that contains the deposits of hydrophobic material that defined the patterned channels and wherein the slide of elastomeric material has been activated so that it can bind to the backing slide, ii) contacting the slide of elastomeric material to which the substrate sheet is adhered to with a backing slide, wherein the backing slide has been activated so that it binds to the slide of elastomeric material at those regions that are not covered by the substrate sheet; wherein the contacting step (ii) is carried out in order to provide sufficient contact for the formation of bonds between the slide of elastomeric material which is not covered with the substrate sheet and the backing slide.

[0159] In a particular embodiment, the slide of elastomeric material is transparent. In another particular embodiment, the backing slide is transparent. In another particular embodiment, the slide of elastomeric material and the backing slide are transparent.

[0160] In a first step, the second method of the invention comprises contacting the slide of the elastomeric material with the side of the substrate sheet, preferably a sheet of filter paper, which is the opposite of the side that contains the deposits of hydrophobic material that defined the patterned channels and wherein the slide of elastomeric material has been activated so that it can bind to the backing slide.

[0161] In a particular embodiment, the slide of elastomeric material is activated by plasma, as a gas plasma or plasma generated by electric current, like sparks. In a preferred embodiment, the slide of elastomeric material is activated by oxygen plasma.

[0162] In another particular embodiment, the backing slide is activated by plasma, as a gas plasma or plasma generated by electric current, like sparks. In a preferred embodiment, the slide of elastomeric material is activated by oxygen plasma.

[0163] Plasma activation is a method of surface modification employing plasma processing, which improves surface adhesion properties of many materials including metals, glass, ceramics, a broad range of polymers and textiles and even natural materials such as wood and seeds. The plasma activation can be performed at atmospheric pressure using air or typical industrial gases including hydrogen, nitrogen and oxygen. Oxygen plasma refers to any plasma treatment performed while introducing oxygen to the plasma chamber. Oxygen is often used to clean surfaces prior to bonding.

[0164] In the second method of the invention, the contacting step (ii) is carried out in order to provide sufficient contact for the formation of bonds between the slide of elastomeric material which is not covered with substrate sheet and the backing slide. In a particular embodiment, the bonds are covalent bonds.

[0165] In a preferred embodiment, the elastomeric material is polydimethylsiloxane (PDMS) and the backing slide is a glass slide. Thus, in a preferred embodiment, after the plasma treatment, the substrate sheet, preferably a sheet of filter paper, were placed over the glass slide and the PDMS was placed on top of it, facing its activated surface towards the non-covered glass surfaces. In a particular embodiment of the second method of the invention, before step i), substrate sheet, preferably a sheet of filter paper, with the pattern of channels is prepared. In a particular embodiment, a laser cutter was used to cut out the paper, and then the the wax is melted into the paper using a hotplate at a temperature between 100°C and 140°C, preferably 120 °C for between 20 to 60 seconds, preferably for 30 seconds.

[0166] In another particular embodiment, the second method of the invention further comprises contacting one or more of the outlets to one or more sensors.

[0167] EXAMPLES

[0168] The following examples illustrate the invention and must not be considered as limiting the scope thereof.

[0169] Material and methods

[0170] Fabrication of the paper-based microfluidic chip

[0171] The developed chip was based on Whatman Grade 1 , 180 pm thickness, (1001-917, Sigma Aldrich, Spain) filter paper. The designs were drawn in Corel Draw Home and Student Suit X7 (Cleverbridge, Spain) and were printed out using a Xerox ColorQube 8580 printer. A laser cutter (VLS2.30DT, Universal Laser Systems, Spain) was used to cut out the paper to precise dimensions, then the wax was melted into the paper using a hotplate at 120 °C for 30 s. The patterned paper substrate was placed between a microscope slide (631-1550, WVR, Spain) and a 26 mm x 76 mm x 2 mm slide of polydimethylsiloxane (PDMS) (Sylgard 184 silicone elastomer 1 :10 kit, Farnell, Spain). For the assembly of the chip, the surfaces of the glass slide and the PDMS slide were activated by oxygen plasma (PDC-002-CE , Harrick plasma, USA) for 30 s (Mitra & Chakraborty, 2011). After activation, the paper channels were placed onto the glass slide before placing the PDMS slide on top of it. The glass and the PDMS bonded to each other between the inlets and outlets after gentle pressure, creating a microfluidic system with several parallel horizontal channels (Ch) (Figure 1A and B).

[0172] The design used in this study consisted of 8 mm wide channels separated by 2 mm wide printed barriers. The width of the channels was chosen so that a sufficient quantity of exudates from the root from the roots are collected by the sensor. It also facilitates acquisition of image data from the sensors and allows to sample the paper itself for further analysis, for example using conventional analytical techniques. The inlets (right hand side of the paper) were 4.0 mm wide and 10.4 mm long, and the outlets (left hand side of the paper) were 4.0 mm wide and 15.8 mm long. The increase length of outlets improved the speed of glucose extraction because evaporation increased with surface area.

[0173] Optimisation and testing of microchannels

[0174] To assess the properties of the flow in the channels blue and yellow food dyes (Sabadell, Vahine, Spain) were diluted 10 times in distilled water. 5 pL of the solution was pipetted to the inlet and pipetting was repeated to maintain saturation of the paper until the liquid reached the outlet part of the channels (Figure 2A). The authors tested the permeability of wax strip of 1 mm, 1 .5 mm and 2 mm width. The dye speed was determined based on the position of the colour front at different time points. The dye was pipetted to the inlet several times (after the dye was completely absorbed by the paper) until the colour front reached the end of the outlets. All tests were carried out in the presence of plant roots.

[0175] Plants

[0176] Two varieties of winter wheat Triticum aestivum (var. rgt Tocayo and var. Filon), were used for the experiments. All seeds were surface sterilised according to O’Callaghan et al. (2018). The seeds were soaked in distilled water for 1 h, then they were immersed into 2 % of calcium hypochlorite (211389, Sigma-Aldrich, Spain) solution for 15 min. Finally, the seeds were rinsed thoroughly using distilled water for a minimum of 10 times in 50 mL. The sterilised seeds were placed onto 0.5 % water agar gel plates (J 10654, Thermo Scientific, Spain) wrapped in aluminium foil at 22 °C for one day. The seedlings were inserted into the microfluidic chips and were incubated for 6 days allowing the roots to reach the bottom channel of the chip. The sensors were prepared just before inserting the seeds into the microfluidic chips to ensure uniform water content across the channels. The samples were kept in a plastic box containing a wet filter paper to maintain constant humidity inside the box. Plants were kept at 22 °C under 14 h light and 10 h dark periods. In order to provide enough replicates, four experimental runs with five replicates were performed with each of the wheat varieties. Each run included two samples without plants as control. Therefore, the total number of samples for each of the wheat varieties was 20 and the total number of the control samples was 8.

[0177] Glucose sensor

[0178] The TNT / alginate hydrogel sensor (Gunatilake et al., 2021, TiO2 Nanotubes Alginate Hydrogel Scaffold for Rapid Sensing of Sweat Biomarkers: Lactate and Glucose. ACS Applied Materials and Interfaces 13: 37734-37745) sensor was polymerised on the top of the paper at the outlet of the paper channels (left side). The sensor is colorimetric. Its initial colour is white and the presence of glucose changes its colour to blue, with the colour intensity relating to glucose concentration. The enzyme complex used for detection consisted of 0.4 mg / mL glucose oxidase (G2133, Sigma-Aldrich, Spain) , 0.05 mg / mL horseradish peroxidase (77332, Sigma-Aldrich, Spain) and 3, 3', 5,5'- tetramethylbenzidine (TMB) (860336, Sigma-Aldrich, Spain) dissolved in dimethyl sulfoxide (> 99.7 %, Sigma-Aldrich, Spain) (24 mg : 2.25 mL ). The enzyme complex was encapsulated in a sodium-alginate (W2011502, Sigma-Aldrich, Spain) hydrogel matrix with additional titanate nanotubes (TNT) (synthetized according to (Gunatilake et al., 2021) ). The TNT content provided a bright white colour for the hydrogel complex, improving the detection of colour changes due to better contrast without modifying the enzymatic reactions.

[0179] Glucose extraction from the microfluidic chips

[0180] After 6 days of incubation, the microfluidic chips with the plants were placed into Petri- dishes (391-0565, VWR, Spain) filled with 40 mL of distilled water. The inlets (right end side) were immersed into the water, while the outlets left facing upward and outside of water (Figure 3D). Water evaporation at the outlet created a gradient in water content, which resulted in a flow of water and exudates to the sensor by capillary action.

[0181] The authors evaluated the time needed for the glucose accumulated in the paper to move through the channel and to be detected by the sensor. Holes with 2 mm of internal diameter were punched into the PDMS (2 mm, Uni-Core punch, Harris, Spain) at the middle of the channels. 1 l of 1mM glucose solution was pipetted into each of the channels and the glucose was extracted using the evaporation. The glucose signals were captured in every two hours for 8 hours in total.

[0182] During experiments measuring glucose concentration in root exudates, flow through the chip were maintained for 2 h before measurement. Images of the sensors were captured using a stereoscope (NSZ-810, Balea Optic, Spain). Once the images were captured, the chips were placed back to the growth box (see Supplementary Information SI-3). Signals from the sensors were collected daily during seven days, and the signal of the six paper microfluidic channels were measured individually. The different sensors were numbered from 1 to 6 respectively from top to bottom (Figure 3D).

[0183] Calibration of the TNT / Alginate sensor for the prediction of glucose concentration in root exudates

[0184] Glucose solutions were prepared in distilled water at a concentration of 0.0, 0.1 , 0.4, 0.8 and 1 mM (G8270, Sigma Aldrich, Spain). Sensors were placed into wax circles (width 1 mm, id. 3mm) on filter paper stripes (Figure 3A). 2 pL of the glucose solution was pipetted on each of the sensors (n = 5). For the control treatment, 2 pL of distilled water were used. To keep the sensors humid, during the chemical reaction, the paper stripes were kept in a sealed Petri-dish and signals from the sensors were captured after 90 min.

[0185] The signals from the sensors were obtained by capturing images using the previously mentioned stereoscope equipped with a digital camera (DS-FI3, Nikon). The images were transformed to HSB images (Hue, Saturation and Brightness) using Imaged software (Imaged). The saturation images were used to identify region of interest, and mean saturation values from circular areas were used as a measure of glucose concentration from the sensor. The relationship between the mean saturation value and the glucose concentration was fitted by the Hill-equation and used to quantify glucose content from the exudates of wheat roots. Since the sugar solution covered the whole surface of the sensors, the whole surface of the sensor was used for the calibration.

[0186] Microfluidic chip fabrication and fluidic testing

[0187] The key elements of the developed wax patterned microfluidic chips are the patterned filter paper, a glass slide, which serves as a substrate and a slice of PDMS on top of the channels. The PDMS is elastic enough to provide space for the seedlings and the roots, and its gas permeability allows enough air for the roots.

[0188] To get the PDMS slice in a reproducible and standard way, a mould was made out of poly(methyl methacrylate) (PMMA) (Goodfellow, Spain) (Figure 6). Using this mould the PDMS slices were always 2 mm thick. One mould had enough space for four PDMS slices. The degassed monomer solution of PDMS (1 :10 initiator: base ratio) was introduced into the mould and it was placed into an oven for 4 h under 60 °C to solidify PDMS. After solidification, the PDMS was removed from the mould and was cut using a scalpel in four individual pieces (26 x 76 x 2 mm) (Figure 6B). These PDMS slices were stored until final fluidic chamber assembly.

[0189] For the chamber assembly, first, the surfaces of the PDMS and the microscope slide were activated by oxygen plasma using the high frequency setting (Harrick plasma, PDC- 002-CE) for 30 s. After plasma treatment, the previously printed, melted and cut paper channels were placed over the glass slide and the PDMS was placed on top of it, facing its activated surface towards the non-covered glass surfaces (Figure 6C). Then, the PDMS was gently pressed to provide enough contact for the formation of the covalent bond between the glass and the PDMS surfaces. The finally assembled microfluidic chip (Figure 6D) was stored until the insertion of the seedlings. Sensor fabrication

[0190] For the sensor preparation, 5 mg of TiC>2 nanotubes were mixed with alginate solution, 1 % in water (Sigma-Aldrich W201502, Spain). The TiC>2 nanotubes create a bright white colour making the detection of colour changes easier. In addition, the nanotubes can also improve the fluidic properties of the sensor. The used enzyme complex, to detect the exuded glucose, consisted of glucose oxidase (GOX, Sigma-Aldrich G2133, Spain) 0.4 mg / mL aqueous solution, horseradish peroxidase (HRP, Sigma-Aldrich 77332, Spain) 0.05 mg / mL in aqueous solution and 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma-Aldrich 860336, Spain) dissolved in dimethyl sulfoxide (DMSO) (> 99.7 %, Sigma-Aldrich, Spain) (24 mg : 2.25 mL). The volume ratio for the hydrogel-enzyme mixture was 30:5:5:5 as alginate-TNT:GOX:HRP:TMB. Using 0.4 M CaCh solution (Sigma-Aldrich 1.02378, Spain), the alginate can be jellified and therefore, integrated into the paper structure.

[0191] For the calibration of the sensor little circles were printed (1 mm line width, ID = 3 mm), and 2 pL of the hydrogel solution with the enzymatic assay was pipetted into each of the circles (Figure 7A). In the next step, in order to initiate the jellification of the alginate, the filter paper was immersed into 0.4 M CaCh solution for 1 min. Then, the paper with the polymerised sensor was rinsed in distilled water for 1 min and let to dry. The devices were fabricated just before the control measurements (Figure 7B) and they were ready to detect glucose solutions (Figure 7C).

[0192] A similar protocol was used for the glucose sensor deposition in the microfluidic chip. In this case, the sensors were placed to the left end of each of the paper outlets without any wax encirclement.

[0193] Collecting means with hexagonal shapes for collecting exudates

[0194] For temporal exudate sampling the following wax printed sampling paper sheet was developed (Figure 8) for the microfluidic device. The sampling paper consist of six hexagonal paper parts for the collection of exudates. Every side of the hexagons are 3.6mm long. The hexagons are connected to a wax printed paper line (Figure 8). This wax printed part holds the hexagons in the desired position and since its hydrophobicity it also prevents the mixing between the liquid content of the hexagons.

[0195] For exudate collection, the hexagons can be inserted to the paper outlet channels through short paper bridges (Figure 9). First, capillary forces in the paper will suck out the liquid samples from the outlet parts of the microfluidic device, later evaporation on the hexagonals will continue this sucking accumulate exudates from the plant root. After the extraction time, the sampling paper pieces can be moved away from the outlets of the chip and the exudates can be dissolved from hexagonal paper part for further analysis using conventional analytical techniques like GC-MS, HPLC etc. This sampling (using new sampling papers) can be repeated as long and as many times as it is desired during the experiment.

[0196] Exudate extraction and analysis

[0197] Aqueous exudates were obtained from the microfluidic device of the invention. Those were further diluted to 30% methanol on to mimic initial composition of chromatographic mobile phase. Five microlitres were injected on a Dionex Ultimate 3000 ultrahigh performance liquid chromatograph (UHPLC, Thermo Fisher Scientific, MA, USA) coupled to a high-performance Q Exactive Focus Orbitrap (q-Orbitrap, Thermo Fisher Scientific) mass analyzer with a heated electrospray ionization source (HESI, Thermo Fisher Scientific), following previously described analytical methods (Phuong K. Nguyen et al., 2020, Analysis of sugars and amino acids in aphid honeydew by hydrophilic interaction liquid chromatography - Mass spectrometry, Methods X, Volume 7, 101050).

[0198] Analyte separation was performed in an ACQUITY UPLC BEH Amide (1.7pm 2.1x150mm) column with pre-column (ACQUITY BEH Amide 1.7pm VANGUARD) from Waters Cromatografia, S.A. Acetonitrile: Milli-Q water (30:70) (A) and Acetonitrile:M illi-Q water (80:20) (B) were used as mobile phase, both containing 0.05 % NH3. Column flow was set at 0.3 mL / min and column was maintained at 35 °C. Gradient elution started with 100 % B during 1 min, arriving to 50 % A at min 10, and then going back to initial conditions at min 11 , and maintained until the end of the injection at min 21 . Before every sequence and every 3 samples and equilibration method of 25 mins at 100% B phase was run for stabilization purposes.

[0199] Measurements were performed in negative ionization in the m / z 50-750 Da range. After a complete scan at a 70000 full width at half maximum (FWHM) resolution, MS2 scans were performed at 17500 FWHM at m / z 200 with an isolation window of 3.0 m / z with a stepped normalized collision energy (NCE) of 30-60-90. The ddMS2 scans were run with an automatic intensity threshold and dynamic exclusion. ACG target was set at 5e4 and its minimum was set at 8.00e3.

[0200] With respect to the HESI parameters, spray voltage was set at 2.5 kV, the capillary temperature was set at 400 °C, the sheath gas at 75 arbitrary units (au), the auxiliary gas at 20 au and 500 °C and the sweep gas at 1 au. Xcalibur 3.1 (Thermo Fischer Scientific) software was used for instrument control and for semiquantitation. A 4 points external calibration with pure standards (Merk) prepared in methanol / water (5-100 pg / L range) was used for the tentative semi quantification.

[0201] Results

[0202] Fabrication of the wax printed patterned paper-based microfluidic chip

[0203] Different chip prototypes were tested in this study, and the design kept for analysis of root exudates consisted of a microscope slide, a wax printed filter paper sheet and a PDMS slide (Figure 1A). The PDMS slide covering the paper channels was an important part of the chip because it is gas permeable but impermeable to water, it kept a humid but aerated environment for the root to grow. The size and shape of printed wax stripes affected the flow of liquids through the paper significantly. Thin wax stripes were not sufficiently impermeable and allowed vertical water flow and mixing between different channels. On the other hand thicker wax stripes occupied large areas and limited the number of sensors in the microfluidic chips. Several stripe widths and channel widths were tested. For width of 1 and 1.5 mm, the printed stripes were permeable and the food dye occasionally mixed between channels. Wax stripes with 2 mm width were found to be impermeable for the duration of all experiments.

[0204] Flow properties in the channels

[0205] The food dyes initially flowed at 0.52 ± 0.05 mm / min in case of the blue dye, and 0.44 ± 0.02 mm / min in the case of yellow dye solutions (Figure 2A and B). After 25 min , the speed slowed down and stabilised at 0.3 ± 0.2 mm / min in the case of the blue dye, and 0.50 ± 0.08 mm / min in case of the yellow dye solutions. The time for the two food dyes to reach the sensing zone of the channels was approximately 35 ± 10 min.

[0206] Calibration curve for the sensor

[0207] The saturation value increased consistently with glucose concentration (Figure 3A and B). The increase was nonlinear and exhibited a plateau for glucose concentration exceeding 1 mM at a saturation value of 117 ± 2 a.u. The relationship was captured by a Hill-equation y = Vmax xn / (kn+xn), where Vmax = 151.71 a.u., k = 0.255 mM, n = 0.929, R2 = 0.9956), and x is the glucose concentration (mM) and y the saturation value in the image (a.u.). The calibration curve allowed the determination of the glucose concentration exuded by the wheat roots (Figure 3B). In the control treatment the signal recorded was equivalent to 0.004 mM glucose. The limit of detection (LOD) was calculated as three times the standard deviation (SD) of the values detected in control treatment, and the limit of quantification (LOQ) was calculated as ten times the SD of the values detected in control treatment. Using these calculations, we obtained a LOD of 0.012 mM and a LOQ of 0.04 mM, respectively.

[0208] Extraction of accumulated glucose from the channels

[0209] Experiments used to characterise the time needed for the sensor to make a measurement showed glucose concentration saturated 4h after a drop of glucose solution was deposited at the inlet (Figure 3C). By comparison, the time of extraction of exudates used on growing plants roots was 2h. The later value was chosen to be sufficient to observe a noticeable increase in the signal acquired from the sensor, but less than the time needed to obtain saturation. This is a necessary compromise to keep the plant growing in vertical position and to allow measurements at sufficient throughput. Therefore, the measured glucose values obtained in growth contained a lag time of 1 day.

[0210] Glucose concentration along the roots

[0211] The authors tested whether the chip can distinguish exudation patterns created from different wheat variety, Triticum aestivum var. rgt Tocayo and var. Filon. The two varieties grew in the paper-based microfluidic chip for 7 days and did not show signs of stress for the duration of the experiment. There was no observable growth differences between the two wheat varieties with mean root lengths for Filon and rgt Tocayo measured at 50.57mm ± 21.4mm and 49.30 ± 21.01 respectively (p=0.73).

[0212] During the experiments, changes in the coloration of the sensors was observed through time (Figure 4). First, the coloration was observed at the base of the root system, at the right hand side of the sensors. With time the sensors became darker and the colouring of the sensor at the bottom of the chip were observed. There were differences in the exudation profile of the two wheat varieties. Glucose was first detected from the rgt Tocayo variety at sensor positions 1 , 2 and 3 during the first days of the experiments (Figure 4A). The detection of the glucose progressed downward during the experiment. In the case of the Filon variety (Figure 4B) colouring appeared 4 days after the start of the experiment. In the control experiments (Figure 4G) no significant colouring of the sensors was observed.

[0213] The quantitative analysis of the signals recorded on the sensors confirmed visual observations (Figure 5 A and B). The distribution and the daily amount of glucose collected by the sensor indicated that the accumulated amount of glucose is higher in the mature part of the root system within the first few days of the experiment. The lower root parts (lower channels) reach their maximum in exudation later in time (day 3 for the rgt Tocayo and day 5-6 for the Filon). The quantity of glucose exuded was significantly different between the two varieties.

[0214] The authors also analysed the exudation rate from the different varieties. Experiments using rgt Tocayo variety showed that at the base of the root system where the concentration of glucose was the highest, the rate of increase of glucose productions was on average 0.035 ± 0.121 mM / day and resulted in more than 0.5 mM glucose deposited on the sensor in the first 4 days of growth. Some samples recorded a total amount of glucose deposited of over 1 mM and approached the saturation zone of the sensor. In the case of the Filon variety, a very low exudation activity was observed at the beginning of the experiment. The exudation started to increase from day 5 and 6, but it was only significant in the first channel (p=0.023), with a maximum glucose production of 0.003 ± 0.037 mM / day.

[0215] Exudate extraction and analysis

[0216] The content of root exudates extracted from the different channels of the developed microfluidic device are showed in Figure 11 . The highest amount of exudates were extracted from the 1st channels and the majority of it was glucose. Glucose was the most abundant exudate component within the 1st, 2nd and 3rd channels. Apart from glucose, D-glucose-6-postphate, maltose monohydrate and glutaric acid gave the major components of the collected root exudates. Apart from these, other molecules in smaller concentration were also observable in the extracted samples (Figure 11).

Claims

CLAIMS1 . A microfluidic device comprising: i) a slide of an elastomeric material; ii) a substrate sheet which contains a pattern of substantially parallel channels, wherein the channels in the pattern are defined by the space between regions in which a hydrophobic material has been deposited onto the substrate; and iii) a backing slide; wherein the substrate sheet is placed between the slide of elastomeric material and the backing slide, wherein the slide of elastomeric material and / or the backing slide is transparent, and wherein the backing slide and the slide of the elastomeric material are bonded to each other at those regions in which the substrate sheet does not separate the two surfaces thereby creating an inlet and an outlet at the opposite ends of each channel in the device.

2. The microfluidic device according to claim 1 wherein the substrate sheet is a sheet of filter paper.

3. The microfluidic device according to claims 1 or 2, wherein the width of the channels are between 250 pm and 1 cm, preferably 8 mm wide.

4. The microfluidic device according to claims 1 to 3, wherein the hydrophobic material is selected from the group consisting of: wax, hydrophobic inks, acrylics and epoxides.

5. The microfluidic device according to claim 4, wherein the hydrophobic material is wax.

6. The microfluidic device according to any of claims 1 to 5, wherein the width of the deposits of the hydrophobic material is of at least 1 ,5 mm, preferably 2 mm.

7. The microfluidic device according to any of claims 1 to 6 wherein the elastomeric material is PDMS.

8. The microfluidic device according to any one claims 1 to 7 wherein the backing slide is transparent.

9. The microfluidic device according to claim 8, wherein the transparent backing slide is a glass slide.

10. The microfluidic device according to any one of claims 1 to 9, wherein the microfluidic device further comprises one or more sensors which are connected to each of the outlets of the device.11 . The microfluidic device according to claim 10, wherein the sensor is selected from the group consisting of: a colorimetric sensor, such as a glucose sensor or a pH sensor, a fluorescent sensor and an electrochemical sensor.

12. The microfluidic device according to claim 11 , wherein the sensor is a glucose sensor.

13. The microfluidic device according to claim 12, wherein the glucose sensor is a colorimetric glucose sensor, preferably an alginate / TiCh hydrogel based glucose sensor.

14. The microfluidic device according to any of claims 1 to 13 wherein the outlets of the channels are connected to collecting means which allow the collection of fluids moving towards the outlet by capillarity.

15. The microfluidic device according to claim 14 wherein the collecting means are made of paper.

16. The microfluidic device according to claims 14 or 15 wherein the collecting means have hexagonal shape.

17. The microfluidic device according to any of claims 14 to 16 wherein the collecting means are connected to the channel outlets by paper connectors.

18. A method for analysing or collecting root exudates and / or root microbiome exudates from different regions within the longitudinal axis of a root, the method comprising: i) inserting a seedling within a device according to any one of claims 1 to 9 under conditions adequate for a root to develop within the substrate sheet in a direction which is substantially perpendicular to the direction of the channels within the device,ii) allowing the root to develop until it reaches in length the position of at least one of the channels in the device and iii) collecting and / or analyzing the exudates from one or more of said channels, simultaneously, after a predefined period of root development.

19. The method according to claim 18 wherein step (iii) is repeated several times at defined time intervals.

20. The method according to claims 18 or 19 wherein the analysis is carried out using a sensor.21 . The method according to claim 20 wherein a sensor is connected to each of the outlets within the device.

22. The method according to claim 21 wherein the incubating step ii) is carried out for a period of time that allows the root of the seedling to reach the channel of the microfluidic device which is opposite to the region in the device wherein the seedling is inserted.

23. The method according to any one of claims 18 to 22, wherein step ii) is carried out for a period of between 7 days and 15 days.

24. The method according to any one of claims 18 to 23 wherein the channel outlets are open so that the exudates move towards the outlet as a result of evaporation of the liquid that pass through the channel open outlets from the inlet.

25. The method according to any one of claims 18 to 24, wherein sensors are connected to one or more outlets in the device.

26. The method according to claim 25, wherein the sensor is selected from the group consisting of: a colorimetric sensor such as a glucose sensor or a pH sensor, a fluorescent sensor and an electrochemical sensor.

27. The method according to claim 26 wherein the sensor is a glucose sensor.

28. The method according to claim 27, wherein the glucose sensor is a colorimetric glucose sensor, preferably is an alginate / TiCh hydrogel based glucose sensor.

29. The method according to claim 18 wherein the outlets of the channels are connected to collecting means which allow the collection of the exudates by capillarity.

30. The method according to claim 29 wherein the collecting means are made of paper.

31. The method according to any of claims 29 or 30 wherein the collecting means have hexagonal shape.

32. The method according to any of claims 29 to 31 wherein the collecting means are connected to the channel outlets by paper connectors.

33. The method according to any one of claims 18 to 32, wherein the seedling is inserted in the microfluidic device at least after one day of germination.

34. The method according to any one of claims 18 to 33, wherein the seedling is from a plant family selected from the group consisting of Solanaceae, Cucurbitaceae, Brassicacea, Poacea and Fabaceae.

35. The method according to any one of claims 18 to 34, wherein the seedling is from a plant genus selected from the group consisting of Arabidopsis, Solanum, Lycium, Citrullus, Cucumis Cucurbita, Lactuca, Brassica, Spinacia, Oryza, Zea, Triticum, Sorghum, Secale, Hordeum, Saccharum, Lens, Cicer, Pisum, Prunus, Pistacia, Citrus, Musa, Vaccinium, Rubus and Olea.

36. The method according to claim 35, wherein the seedling is from the genus Triticum, preferably from the species Triticum aestivum.

37. Use of the microfluidic device according to any one of claims 1 to 17 for spatial and / or temporal detection of root exudates and / or root microbiome exudates.

38. A method for the manufacture of the microfluidic device according to any of claims 1 to 17 comprising: i) contacting the slide of the elastomeric material with the side of the substrate sheet which is the opposite of the side that contains the deposits of hydrophobic material that defined the patterned channels and wherein the slide of elastomeric material has been activated so that it can bind to the backing slide,ii) contacting the slide of elastomeric material to which the substrate sheet is adhered to with a backing slide, wherein the backing slide has been activated so that it binds to the slide of elastomeric material at those regions that are not covered by the substrate sheet; wherein the contacting step (ii) is carried out in order to provide sufficient contact for the formation of bonds between the slide of elastomeric material which is not covered with the substrate sheet and the backing slide.

39. The method according to claim 38 further comprising contacting one or more of the outlets to one or more sensors.

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