Cellulose based devices for microorganism detection, methods and uses thereof

Abstract

The present disclosure relates to the field of microbiological analysis using a miniaturized cellulose-based microbiological culture device and a method to produce the same. Particularly to a device designed to detect microorganisms in various samples using a specially configured cellulose substrate.

Description

D E S C R I P T I O NCELLULOSE BASED DEVICES FOR MICROORGANISM DETECTION, METHODS AND USES THEREOFTECHN ICAL FIELD

[0001] The present disclosure relates to the field of microbiological analysis using a miniaturized cellulose-based microbiological culture device and a method to produce the same. Particularly to a device designed to detect microorganisms in various samples using a specially configured cellulose substrate.BACKGROUND

[0002] Microbiological contamination analysis is one of the key areas in the food and beverage industries. Used routinely for the identification and characterization of microorganisms, it is a valuable tool to access safe production, transportation, and storage of food products as well as to evaluate the effectiveness of cleaning procedures and safety protocols within the industry.

[0003] While standard bacterial culture remains the gold standard technique, several technologies have been introduced to facilitate detection procedures. Standard microbiology laboratory procedures still rely on liquid media or solid culture media in a Petri dish format, with agar being the most common gelling agent used in the production of solid culture media. However, most of these have low shelf life (weeks) and require refrigeration during transport and storage (Lagier et al., 2015). Moreover, these techniques were developed by and for microbiology laboratories and specialized technicians. The present disclosure was developed with several principles focusing on bringing microbiological contamination detection procedures outside the laboratory to be able to be used in the open world alongside produce during its life cycle: from production and shipment to storage.

[0004] The market leader for agar Petri dish alternatives is the 3M PETRIFILM technology. These devices are meant to improve efficiency over conventional Petri dish plating, due to their ability to be stored dry and its low thickness, which allow for improved stacking. 3M PETRIFILM is generally comprised of a cold-water soluble hydrogel forming polymers such as guar gum, xanthan gum, alginate, carboxymethyl cellulose, hydroxyethyl cellulose, etc., nutrients for microbiological growth and a chromogenic indicator to detect a specific microorganism species or strain. Several patents of the same technical field of the present disclosure have been issued, including, but not limited to the documents US20200056136A1, US9873904B2 and WO2012092242A2. US20200056136A1 discloses a thin-film device for detection of microorganisms.

[0005] Document US20230390761A1 discloses a card device for detecting the existence and / or identity of microbial presence, having a base for receiving thereon a fluidic sample, a transparent cover to overlay the base, formed with a top layer, a bottom layer, and an adhesive layer therebetween, wherein a portion of the bottom layer is formed such that a portion of the adhesive layer is not covered by the bottom layer, and a media is applied to and retained by that portion of the adhesive layer which is not covered by the bottom layer, that media serving to gel the fluidic sample and / or support growth of a microorganism whose existence and / or identity is being tested by the card. The present disclosure differs from this document in the sense that culture media for microbial growth / detection is applied to and retained in a cellulose-based hydrogel layer, arranged on top of or partially integrated on the cellulose substrate layer.

[0006] Document US20230390761A1 is continued-in-part by document US20230390775A1, entitled "Improved Microbiology Test Card", which discloses a test card for separate but simultaneous or sequential detection of the existence and / or identity of different samples of microorganisms, having three layers which allow total separation, growth, and testing of different test samples which require chemically different growth media or replicative simultaneous testing. In the disclosed document, it is stated that all layers should be non-water absorbent, contrary to the present invention, where the bottom layer is a water absorbent, cellulose fibrous layer and the detection layer is a hydratable hydrogel, where the microorganisms can grow, and that can be impregnated with specific colorimetric media for microbial detection. Thus, the present disclosure leverages the properties of its composing material's matrix to transport, collect, grow, and detect microorganisms. Another aspect which distinguishes the two technologies lies in the simultaneous detection of distinct microorganisms in the same device. In document US20230390775A1, only two different samples, growth media or conditions can be tested in the same device, one on each side of the card.

[0007] Document W02021170606A1 discloses a ready-to-use device and method for testing differential growth of microorganisms. The device comprises a base sheet, a dry medium layer on top of the base sheet, a water vapor impermeable lid sheet to cover the medium layer and one or more differentiating agents are positioned under, within, on and / or above the dry medium layer in a spatially resolved manner. The present disclosure differs from this document in its materials, being composed of several cellulose or cellulose-based layers. Particularly, the bottom layer is composed of hydrophilic paper to induce absorption of liquid samples and transport them into the sensing hydrogel layer Document W02021170606A1 discloses a technology that is focused on differential growth profiles mainly used for antibiogram / E-test like devices instead of multiple independent specific and chromogenic media for bacterial identification. Several patent documents disclose planar or layered culture devices that employ structured substrates and are embedded with reagents for the analysis biological samples.

[0008] Document WO2017027956A1 discloses an apparatus, method, and hydrogel for testing for the presence of pathogens in test liquids.

[0009] Document GB2619127A describes a device for detecting an analyte in a fluid sample, the device comprising a testing element and a housing accommodating the testing element, wherein the housing is formed by folding a paper material. The test element comprises a test area and a sample application area which are connected such that the fluid sample travels from the sample application area to the test area by wicking or capillary action.

[0010] US20110105360A1 discloses three-dimensional cellular arrays, methods of making said arrays, and methods of identifying bioactive agents, namely, drugs, for use in high-throughput screening employing eukaryotic cells.

[0011] US10704078B2 discloses a layered device and method for isolating microorganisms from a sample on a culture medium.

[0012] US20150010941A1 discloses a thin-film culture devices incorporating nutrient media, pH indicators, and selective agents for detecting acid-producing bacteria.

[0013] US20230349008A1 sample testing chip with patterned hydrophobic barriers.

[0014] Rapid and reliable detection of microorganisms in environmental, biological, or clinical samples is crucial for various applications including healthcare, quality control in food and beverage industries, and environmental monitoring. Traditional methods typically require complex lab equipment and lengthy cultivation times to identify contamination. Thus, there is a need for a simple, efficient, and portable device that can quickly detect microorganisms in samples.

[0015] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GEN ERAL DESCRIPTION

[0016] The present disclosure addresses the aforementioned needs by providing a device for detecting a microorganism in a sample. The device, namely a tag, comprises a cellulose substrate with multiple cavities, which serve as testing zones or spots, each cavity containing a composite layer. This composite layer includes a rehydratable hydrogel and a culture medium constructed for cultivating a microorganism. The substrate was developed to absorb the sample through capillary action directly to the bottom of the cavities, facilitating immediate interaction between the sample and the composite layer. The present invention also relates to the use of the cellulose-based microbiological culture device for culture, detection, characterization, identification, and enumeration of microorganisms.

[0017] An aspect of the present disclosure relates to a device for detecting a microorganism in a sample comprising a cellulose substrate with a plurality of cavities; a composite layer within each cavity, comprising a rehydratable hydrogel and a culture medium for cultivating a microorganism; the cellulose substrate is configured to absorb the sample via capillary action, preferably into the bottom of the cavities, to facilitate interaction between the sample and the composite layer for the detection of a microorganism in the sample. The capillary-based inoculation enables hands-free sample transfer, which eliminates the need for pipettes or droppers, supporting the usage in the field and by non-trained personnel.

[0018] Another aspect of the present disclosure relates to the device for detecting a microorganism in a sample, comprising a cellulose substrate; a composite layer on the top of the cellulose substrate comprising a rehydratable hydrogel and a culture medium for cultivating a microorganism in the top of the cellulose substrate; the cellulose substrate is configured to absorb the sample via capillary action to facilitate interaction between the sample and the composite layer for the detection of a microorganism in the sample; the cellulose substrate surrounding the composite layer comprises at least one hydrophobic compound or a mixture of such compounds to prevent the spread of the sample and culture medium outside the composite layer.

[0019] Surprisingly, the present disclosure provides a simplified, efficient, and cost-effective method for detecting microorganisms, enhancing the capabilities of environmental, clinical, and food and beverages safety testing. The present disclosure facilitates rapid and direct interaction between the sample and the culture medium, reducing detection times. The device's simple design and use of a paper substrate make it lightweight, disposable, and easy to handle in various settings.

[0020] The present disclosure exhibits several integrated innovative solutions, such as:1) long shelf life at room temperature by means of stabilization of reagents in a dehydrated natural polymer matrix that is rehydratable before use;2) multi layered miniaturized design: unlike Petri dishes, this device features a multilayered laminated structure. The inclusion of low and high-water absorption cellulose layers ensures efficient moisture management. These layers maintain optimal hydration for microbial growth, promoting homogeneous color change, hence accurate results;3) multiple discrete detection cavities, namely zones or spots: Within the device, a discrete detection zone allows for multiple growth media reactions, enabling characterization and identification of microorganisms. Whether it's bacterial species or fungal strains, a single or multi spot (or cavity) device can provide comprehensive characterization insights by using colorimetric and chromogenic reagents. Moreover, the reduction in size and multiplex capabilities allow to dramatically reduce reagents;4) self-inoculating capability and integrated colorimetric reactions: the cellulose-based device leverages paper's natural hydrophilicity and passive fluidics. It can self-inoculate without specialized equipment. This eliminates the need for complex laboratory setups, making it ideal for fieldwork or resource-limited settings; coupled with colorimetric and chromogenic reactions this system allow for easy score of results;5) high volume sample analysis; the encapsulation in a hard polymer casing, namely involucre, further increases the application making it capable of analyzing high volume samples. This characteristic is particularly important in clinical setting (e.g. urine analysis); food and beverage industry (high volume samples (> 3mL) with high dilution factor of bacterial contamination), and or environmental monitoring (water bodies and affluents safety monitoring);6) renewable and biodegradable materials: the composition of the device relies on bio-based and biodegradable materials. By using cellulose, a product that is mostly based on natural occurring polymers, the environmental footprint is reduced;7) automated quantitative analysis capabilities: automated imaging systems and analysis algorithms can easily record and score colorimetric changes. This integration streamlines data collection and analysis, enhancing efficiency while introducing quantitative capabilities to the present disclosure.

[0021] The present disclosure makes innovative use of cellulose in multiple roles: as a wicking, watermanaging fibrous substrate; as the base structure for supporting hydrogel layers; and as part of a multilayered architecture designed to control hydration and sample movement. The hydrogel itself comprises cellulose-derived hydrocolloids such as carboxymethyl cellulose (CMC) and other copolymers, optimized for complete dehydration and room-temperature rehydration. This results in a device that is not only biodegradable and low-cost, but also room temperature stable for long-term storage and effective in non-laboratory environments.

[0022] In an embodiment for better results, the device comprises multiple layers (multi-layer laminated structure) with differing water absorption properties ( / .e., varying hydrophilicities) to actively regulate sample hydration and hydrogel rehydration, thereby optimizing microbial growth.

[0023] In an embodiment for better results, the cellulose substrate surrounding the composite layer may comprise at least one hydrophobic compound or a mixture of such compounds to prevent the spread of the sample and culture medium outside the composite layer.

[0024] In an embodiment for better results, the top layer may be encapsulated by an involucre comprising a water impermeable polymer.

[0025] In an embodiment for better results, the number of the plurality of cavities may be 2 - 50 cavities, preferably 3 - 40 cavities; more preferably 4 - 30 cavities; even more preferably 5-20 cavities. Namely: 2, 3, 4, 5, 6, 7, 8, 9 or 10 cavities.

[0026] In an embodiment for better results, the cellulose substrate is a cellulose fibrous layer.

[0027] In an embodiment for better results, the cellulose substrate comprises a plurality of layers.

[0028] In an embodiment for better results, the plurality of layers of the cellulose substrate ranges from 2-10 layers; preferably 2-5 layers, more preferably 3-5.

[0029] In an embodiment for better results, the cellulose substrate is paper.

[0030] In an embodiment for better results, the cellulose substrate is selected from the group consisting of: bleached softwood fibers, unbleached softwood fibers, bleached hardwood fibers, unbleached hardwood fibers, cotton fibers, or mixtures thereof.

[0031] In an embodiment for better results, the cellulose substrate grammage (expressed in grams (g) per square meter (m2)) ranges from 50 g / m2- 500 g / m2; preferably from 100 g / m2- 400 g / m2; more preferably from 200 g / m2- 300 g / m2.

[0032] In an embodiment for better results, the thickness of the cellulose substrate ranges from 300 pm - 1 mm, preferably 350 pm - 600 pm; more preferably 400 pm - 500 pm.

[0033] In an embodiment for better results, the Schopper degree (°SR) of the cellulose substrate ranges from 15 °SR - 40 °SR at 20 °C. The Schopper degree (°SR), also known as the Schopper-Riegler degree, is a unit of measurement used to quantify the drainability or freeness of pulp suspensions in the paper industry. This parameter provides an indication of how easily water can be removed from the pulp. It was measured in a Schopper-Riegler apparatus at 20 °C.

[0034] In an embodiment for better results, the cellulose substrate can have fiber in low consistency (2 to 6%), medium consistency (6 to 15%) or high consistency (15 to 40%) with final Schopper Riegler freeness degree (°SR) between 15 and 40.

[0035] In an embodiment for better results, the cellulose substrate further comprises at least one cross-linker agent and / or at least one wet strength agent in order to bound composite layer to the substrate.

[0036] In an embodiment for better results, the cross-linker agent of the cellulose substrate ranges from 0.01% weight (wt.) - 5% wt.; preferably 0.05 wt. - 2% wt., more preferably 0.1 wt.- 1% wt.

[0037] In an embodiment for better results, the cross-linker agent of the cellulose substrate is selected from the group consisting of: zinc chloride, magnesium chloride, copper sulfate, nickel sulfate, calcium chloride, epichlorohydrin, or citric acid, or mixtures thereof.

[0038] In an embodiment for better results, the wet strength agent of the cellulose substrate is selected from the group consisting of: polyaminopolyamide-epichlorohydrin resins, glyoxalated polyacrylamide resins, polyvinylamine resins, polyethylenimine resins, polyisocyanate resins, dialdehyde starch, or mixtures thereof.

[0039] In an embodiment for better results, the composite layer is impregnated or coated.

[0040] In an embodiment for better results, the composite layer comprises a plurality of sub-layers.

[0041] In an embodiment for better results, the composite layer comprises at least 1 to 4 sub-layers.

[0042] In an embodiment for better results, the composite layer is arranged on top of or partially integrated in the cellulose substrate.

[0043] In an embodiment for better results, the composite layer is partially integrated in the cellulose substrate, forming an interface layer that enables the anchorage of the rehydratable hydrogel.

[0044] In an embodiment for better results, the interface layer has a thickness between 0.1 pm - 500 pm; preferably 1 pm - 100 pm, more preferably 5 pm - 50 pm.

[0045] In an embodiment, the composite layer thickness ranges from 5 pm - 500 pm; preferably 10 pm - 300 pm, more preferably 20 pm - 200 pm.

[0046] In an embodiment for better results, the device of the present disclosure comprises a plurality of perforations in at least one of the layers or sublayers of the substrate and / or the composite layer.

[0047] In an embodiment for better results, the hydrogel grammage in the substrate ranges from 20 g / m2- 600 g / m2; preferably 50 g / m2- 400 g / m2, more preferably 100 g / m2- 300 g / m2.

[0048] In an embodiment for better results, the amount of culture medium in the composite layer ranges from 5 g / m2- 500 g / m2; preferably 20 g / m2- 400 g / m2, more preferably 30 g / m2- 300 g / m2.

[0049] In an embodiment for better results, the composite layer comprises at least one binding agent, one gelling agent, and one plasticizer, or mixtures thereof.

[0050] In an embodiment for better results, the binding agent of the composite layer ranges from 0.1% wt. - 6% wt.; preferably 0.5% wt. - 5% wt., more preferably 1% wt. - 4% wt.

[0051] In an embodiment for better results, the binding agent of the composite layer is a cellulosederived hydrocolloid; preferably cellulose and / or a cellulose derivative, a polynucleotide, a polypeptide, a polysaccharide, a natural rubber, a polyphenolic polymer, or a complex of polymers of large chain fatty acids, or mixtures thereof.

[0052] In an embodiment for better results, the binding agent of the composite layer is selected from the group consisting of: sodium carboxymethyl cellulose (CMC), carboxyethyl cellulose (CEC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), ethyl cellulose (EC), hydroxypropylmethyl cellulose (HPMC), hydroxyethylmethyl cellulose (HEMC), hydroxyethylpropyl cellulose (HEPC), bacterial cellulose (BC), cellulose nanofiber (CNF), cellulose nanocrystals (CNC), microfibrillated cellulose (MFC), hydroxypropyl methylcellulose phthalate, or mixtures thereof; preferably carboxymethyl cellulose, sodium carboxymethyl cellulose, or mixtures thereof.

[0053] In an embodiment for better results, the gelling agent of the composite layer ranges from 0.1% wt. - 6% wt.; preferably 0.5% wt. - 5% wt., more preferably 1% wt. - 4% wt.

[0054] In an embodiment for better results the gelling agent of the composite layer is a polysaccharide hydrocolloid.

[0055] In an embodiment for better results, the gelling agent of the composite layer is selected from the group consisting of: sodium alginate, gellan gum, carrageenan, guar gum, xanthan gum, locust bean gum, gum arabic, pectin, modified starch, or mixtures thereof.

[0056] In an embodiment for better results, the plasticizer of the composite layer ranges from 1% wt. - 15% wt.; preferably 2% wt. - 12% wt., more preferably 3% wt. - 8% wt.

[0057] In an embodiment for better results, the plasticizer of the composite layer is selected from the group consisting of: polyvinyl alcohol (PVOH), ethylene glycol (EG), diethylene glycol (DEG), triethylene glycol (TEG), polyethylene glycol (PEG), propylene glycol (PG), glycerol, erythritol, sorbitol, mannitol, maltitol, xylitol, polyols, fatty acids, vegetal oils, or mixtures thereof.

[0058] In an embodiment for better results, the culture medium of the composite layer in each cavity is different to enhance specificity for different microorganisms.

[0059] In an embodiment for better results, the culture medium of the composite layer in each cavity comprises variations in the composition or different concentrations to enhance specificity for the detection of different microorganisms.

[0060] In an embodiment for better results, the composite layer may further comprise antimicrobial agent; preferably an antibiotic to suppress the growth of non-target microflora and for the identification of the microorganism resistance.

[0061] In an embodiment for better results, the amount of antimicrobial agent in each cavity may be different to test susceptibility of microorganisms to different antimicrobial agent or to a different concentration of the same antimicrobial agent.

[0062] In an embodiment for better results, the cavity, preferably a plurality of cavities, in the cellulose substrate layer has a gradient of antimicrobial agent concentration (for example E-test, Fig. 5 and Fig. 24).

[0063] In an embodiment for better results, the composite layer further comprises a dye and / or chromogenic agent that changes color in the presence of a target microorganism, thereby indicating the presence and potentially the concentration of the microorganism in the sample.

[0064] In an embodiment for better results, the composite layer further comprises at least one cell growth marker.

[0065] In an embodiment for better results, the hydrophobic compound of the device of the present disclosure further comprises at least one transparent and hydrophobic polymer.

[0066] In an embodiment for better results, the hydrophobic compound is selected from the group consisting of: ethyl cellulose, cellulose acetate, cellulose acetate butyrate (CAB), cellulose propionate, cellulose oleate, cellulose laurate, silylated cellulose, or cellulose acetate phthalate (CAP), or mixtures thereof.

[0067] In an embodiment for better results, the encapsulation involucre further comprises a polymer.

[0068] In an embodiment for better results, the polymer is a flexible polymer and / or a hard polymer.

[0069] In an embodiment for better results, the flexible polymer is selected from a list consisting of: polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PU), silicone rubber, thermoplastic elastomers (TPE), polydimethylsiloxane (PDMS), polycarbonate (PC), nylon, polyamide, ethylene vinyl acetate (EVA), polyvinylidene fluoride (PVDF), ethylene propylene diene monomer (EPDM), natural rubber (NR), styrene-butadiene rubber (SBR), or polyisoprene, or mixtures thereof.

[0070] In an embodiment for better results, the hard polymer is selected from a list consisting of: polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyethylene terephthalate (PET), polypropylene (PP), acrylonitrile butadiene styrene (ABS), polyetheretherketone (PEEK), polyoxymethylene (POM), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyphenylene sulfide (PPS), polyvinylidene fluoride (PVDF), or polyamide, nylon, or mixtures thereof.

[0071] In an embodiment for better results, the device of the present disclosure may further comprise a handling zone for the transportation or placement of the said device.

[0072] In an embodiment for better results, the handling zone may be connected to rehydration means in order to avoid the contamination of the sample.

[0073] Another aspect of the present disclosure relates to a kit for detecting a microorganism in a sample comprising the device of the present disclosure, preferably wherein said kit further comprises a container for the sample.

[0074] Another aspect of the present disclosure relates to a method for producing the device of the present disclosure for detecting a microorganism in a sample, comprises the steps of: impregnating a cross-linker agent in the cellulose substrate; applying a rehydratable hydrogel in the cellulose substrate according to the number of rehydratable hydrogel layers desired, forming at least one hydrogel layer; drying the said device at a temperature between 20° degrees Celsius (°C) - 50 °C; washing the said device in water for a period inferior to 20 minutes; adding one or several culture media solutions to each testing cavities; drying the said device at a temperature between 20 - 50 °C; individualization ofthe cavities and removal of hydrogel in unnecessary areas; adding a culture medium solution to the rehydratable hydrogel individualized cavities; drying the device at a temperature between 20 - 50 °C; and, lamination of all the device layers.

[0075] In an embodiment for better results, the method for producing the device of the present disclosure for detecting a microorganism in a sample further comprises depositing at least one antibiotic gradient solution and a colorimetric agent in the composite layer.

[0076] In an embodiment for better results, the method for producing the device of the present disclosure for detecting a microorganism in a sample further comprises the lyophilization of the hydrogel as the last step.

[0077] Another aspect of the present disclosure relates to the use of the device of the present disclosure for the culture, growth, detection, characterization, identification, and enumeration of microorganisms.

[0078] In an embodiment for better results, the use of the device of the present disclosure with biological, food, beverage or environmental samples.

[0079] In an embodiment for better results, the use of the device of the present disclosure comprises contact inoculation by direct contact with a surface.

[0080] In an embodiment for better results, the use of the device of the present disclosure comprises inoculation via passive capillarity using an inoculation strip.

[0081] In an embodiment for better results, a method for using the device of the present disclosure may further comprise placing the said device under a camera apparatus for real-time color monitoring.BRIEF DESCRIPTION OF TH E DRAWI NGS

[0082] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the present disclosure.

[0083] Figure 1: Schematic representation of an embodiment of a single spot or cavity (inoculation strip) both top view and cross section view of the device.

[0084] Figure 2: Schematic representation of an embodiment of a multi spot or cavity (inoculation strip) both top view and cross section view of the device.

[0085] Figure 3: Schematic representation of an embodiment of a multi spot or cavity (drop inoculation) both top view and cross section view of the device.

[0086] Figure 4: Schematic representation of an embodiment of a multi spot or cavity (drop-inoculation for high volume cup) both top view and cross section view of the device.

[0087] Figure 5: Schematic representation of an embodiment of E-test device architecture (drop inoculation) both top view and cross section view of the device.

[0088] Figure 6.1: Schematic representation of an embodiment of encapsulation, soft-shell stick-on adhesive.

[0089] Figure 6.2: Schematic representation of an embodiment of a removable sheet to protect adhesive before placement.

[0090] Figure 7.1: Schematic representation of an embodiment of encapsulation; hard-shell (capillary inoculation) both top view and cross section view of the device.

[0091] Figure 7.2: Schematic representation of an embodiment of encapsulation; hard-shell (direct contact inoculation) both top view and cross section view of the device.

[0092] Figure 7.3: Schematic representation of an embodiment of encapsulation; hard-shell cup (large sample volumes) both top view and cross section view of the device.

[0093] Figure 8 - Soft-shell encapsulation of the device of the present disclosure. Top) Front, side and back views. Bottom) of the device of the present disclosure with soft shell encapsulation glued onto a typical household plastic container.

[0094] Figure 9 - 3D printed prototypes of hard-shell encapsulation of the device of the present disclosure. Left - capillary inoculation; right - direct contact inoculation.

[0095] Figure 10 - Hard-shell cup for large sample volume inoculation of the device of the present disclosure.

[0096] Figure 11 - Single spot (or cavity) devices with different culture media after 24h incubation with Enterococcus faecal is.

[0097] Figure 12 - Single spot (or cavity) devices with different culture media and inoculated with distinct bacterial species.

[0098] Figure 13 - Multi spot (or cavity) devices with different culture media inoculated with distinct bacterial species.

[0099] Figure 14 - Testing of the device of the present disclosure in fish (top) and inside wall of box (bottom), left to spoil at ambient temperature for 14 days (left - day 0; right - day 14).

[0100] Figure 15 - Close up of the device of the present disclosure. Left - Devices with distinct culture media all placed directly on top of fish. Right - Devices with the same culture media (Enterococci ChromoSelect Broth) placed in different locations.

[0101] Figure 16 - Testing of the device of the present disclosure in spoiling fish. Daily evolution of color.

[0102] Figure 17 - Bacterial growth curves obtained by RGB analysis (RGB color system) of the device of the present disclosure.

[0103] Figure 18 - Bacterial growth curves obtained from RGB analysis of the device of the present disclosure on spiling fish. Median values, combining all the different culture media used, per device location.

[0104] Figure 19 - Single spot (or cavity) devices on spoiling fish tests.

[0105] Figure 20 - Multi spot (or cavities) devices on spoiling fish tests

[0106] Figure 21 - Device of the present disclosure with Enterococci ChromoSelect Broth exposed to Enterococcus faecalis at different bacterial load for 18h. Dilution factor MacFarland standard (from left to right: 0; IO6; 10s; IO4; IO3).

[0107] Figure 22 - Variation of color intensity with time at different bacteria dilution factors (RGB analysis).

[0108] Figure 23 - Variation of curve slope with dilution factor. Slope obtained from trendline of growth phase from color intensity / time graph.

[0109] Figure 24 - E-test-like device with chromogenic culture media and antibiotic gradient, after bacterial growth.DETAILED DESCRIPTION

[0110] The present disclosure relates to the field of microbiological analysis using a miniaturized cellulose-based microbiological culture device and a method to produce the same. Particularly to a device designed to detect microorganisms in various samples using a specially configured cellulose substrate.

[0111] The present disclosure pertains to the field of microbiological analysis. Specifically, it describes a cellulose-based microbiological detection device. The device of the present disclosure comprises a multilayered structure with both low and high-water absorption cellulose layers and a composite layer containing a rehydratable hydrogel combined with a suitable culture medium for microorganism detection. The culture medium preferably includes a hydrophobic compound or a mixture of such compounds, and / or a watertight encapsulation material. Additionally, the disclosure presents a cellulose-based microbiological culture device with cavities, such as testing zones or spots, enabling multiple and simultaneous growth media reactions. This design facilitates the detection, characterization, and identification of various microorganisms within a single device.

[0112] In an embodiment, the device comprises a plurality of cavities, namely testing zones or spots, each infused with a distinct culture medium and indicator, thereby enabling the simultaneous detection of different microorganisms and / or the evaluation of different conditions from the same sample.

[0113] In an embodiment, the cellulose-based microbiological culture device comprises the following layers as seen in Figures 1 to 7:- a cellulose substrate (1) to allow for homogeneous water absorption;- a composite layer comprising a rehydratable hydrogel and a culture medium (2) arranged on top of or partially integrated on the cellulose substrate (1);- a hydrophobic compound or a mixture of such compounds to be used as a spacer to allow hydrogel swelling without compression (3); at least one encapsulation layer (4) comprising a water impermeable polymer to allow for incubation at optimal bacterial growth temperatures with limited water evaporation; wherein the polymer can be flexible or nonflexible allowing for a flexible stick-on surface device or a hard case encapsulation for high sample volume applications;- wherein the rehydratable hydrogel of the composite layer (2) comprises: at least one binding agent from the family of cellulose-derived hydrocolloids in a concentration between 0.1% wt. and 6% wt.; at least one gelling agent from the family of polysaccharide hydrocolloids in a concentration between 0.1% wt. and 6% wt.; and a plasticizer in a concentration between 1% wt. and 15% wt.; and wherein the at least one cellulose substrate layer (1) further comprises a cross-linker agent in a concentration between 0.01% wt. to 5% wt. At least one encapsulation layer; wherein the encapsulation can be flexible or non-flexible polymers allowing for a flexible stick-on surface device or a hard case encapsulation for high sample volume applications.

[0114] In the context of the present disclosure, in a particular embodiment, "partially integrated on" means that a portion of the rehydratable hydrogel of the composite layer (2) is impregnated in the cellulose substrate (1), creating a boundary section wherein the hydrogel material is mixed with the cellulose substrate material. The interface wherein the hydrogel material and the cellulose substrate material combine has a thickness between 0.1 pm and 500 pm. This partial integration assures a controlled expansion of the hydrogel in the desired direction upon hydration and prevents detachment of the rehydratable hydrogel from the cellulose substrate layer.

[0115] In the context of the present disclosure, in a particular embodiment, "arranged on top of" means that the rehydratable hydrogel of the composite layer (2) is bound to the cellulose substrate layer (1) without creating an interface of combined materials.The cellulose substrate layer (1):

[0116] In an embodiment, the cellulose-based microbiological culture device of the present disclosure comprises a cellulose substrate layer (1) comprising cellulose fibers with grammage in the range of 50 g / m2to 500 g / m2. The cellulose fibers can be selected from different sources, including but not limited to, bleached softwood fibers, unbleached softwood fibers, bleached hardwood fibers, unbleached hardwood fibers, cotton fibers, or any other suitable fiber source known for a person skilled in the art or mixtures thereof. Additionally, different processes can be used to obtain said fibers, including but not limited to sulfite pulping, kraft pulping, general chemical pulping, mechanical pulping, chemi-mechanical pulping, or any other pulping process known to a person skilled in the art.

[0117] In an embodiment, the cellulose substrate layer (1) is obtained from refining the fibers until the desired Schopper Riegler freeness degree (°SR). Refined fibers have a °SR between 15 and 40. Fibers with different Schopper Riegler freeness degree can be combined to obtain a cellulose substrate layer with the appropriated water retention capability. Fiber refining can be done in low consistency, medium consistency or high consistency using processes known to a person skilled in art, including but not limited to conical refiners or disc refiners.

[0118] In an embodiment, at least one cellulose substrate layer (1) comprises a cross-linker agent. The cellulose substrate layer (1) comprising the cross-linker is preferably the layer directly in contact with the rehydratable hydrogel of the composite layer (2).

[0119] In an embodiment, the cellulose substrate layer (1) is used as substrate for the rehydratable hydrogel of the composite layer (2) but also as a cross-linker diffusion matrix allowing gelation to occur only at the time of contact between the polymers and the substrate, this allows the hydrogel formulation to be viable for a much longer period, since cross-linking does not occur immediately in the mixing process of the components of the device. The second advantage is achieving a homogeneous distribution of the cross-linker along the cellulose substrate layer (1). The third advantage is that it prevents all the hydrogel from entering the cellulose substrate layer (1) as the gelling occurs on the two layers interface, allowing the vast majority of the cellulose substrate layer (1) volume to remain with the original structure while maintaining a high rehydration speed and water retention capacity. In an embodiment, the cross-linker agent improves hydrogel strength and is selected from, but not limited to, zinc chloride, magnesium chloride, copper sulfate, nickel sulfate, calcium chloride, epichlorohydrin, or citric acid.

[0120] In an embodiment, the cross-linker agent is present between 0.01% wt. and 5% wt. of hydrogel before drying, preferably between 0.05% wt. and 2% wt., more preferably between 0.1% wt. and 1% wt.

[0121] In an embodiment, the cellulose substrate layer (1) contains a wet strength agent selected from, but not limited to, polyaminopolyamide-epichlorohydrin resins, glyoxalated polyacrylamide resins, polyvinylamine resins, polyethylenimine resins, polyisocyanate resins, dialdehyde starch, or mixtures thereof.

[0122] In an embodiment, the cellulose-based microbiological culture device comprises at least two stacked cellulose substrate layers allowing to increase the water retention capabilities. In this embodiment, the lower cellulose substrate layer, which is not in direct contact with the hydrogel, is arranged and bonded below the upper cellulose substrate layer. The first cellulose substrate layer allows the hydration of the device from the bottom.The composite layer comprising a rehydratable hydrogel and a culture medium (2):

[0123] In an embodiment, the rehydratable hydrogel of the composite layer (2) comprises:- at least one binding agent from the family of cellulose-derived hydrocolloids;- at least one gelling agent from the family of polysaccharide hydrocolloids;- a plasticizer.- At least one growth media per test spot with colorimetric and / or chromogenic indicator.

[0124] In an embodiment, the number of layers of the composite layer (2) present on the cellulose substrate layer (1) varies from 1 to 4.

[0125] In an embodiment, the composite layer (2) has a thickness between 5 pm and 500 pm, preferably between 20 pm and 200 pm.

[0126] In an embodiment, the rehydratable hydrogel has a grammage between 20 g / m2and 600 g / m2, preferably between 100 g / m2and 300 g / m2.

[0127] In an embodiment, the rehydratable hydrogel covers the entire surface of the device - devices with one single test spot (or cavity).

[0128] In an embodiment, the hydrogel is individualized into multiple independent test spots (or cavities) or specific zones allowing for the integration of multiple and independent growth media for specific detection and identification of microorganisms.

[0129] In an embodiment, contiguous test spots can be impregnated with increasing quantities of specific antibiotics producing a device for antibiotic susceptibility testing (E-test) (Fig. 24).

[0130] In an embodiment, several hydrogel polymers may be selected according to their sustainable features regarding physical and chemical characteristics that promote the desired cellular growth while allowing for full dehydration and further rehydration steps.

[0131] In an embodiment, the hydrocolloid binding agent for the rehydratable hydrogel is selected from the group comprising cellulose and / or a cellulose derivative, a polynucleotide, a polypeptide, a polysaccharide, a natural rubber, a polyphenolic polymer, a complex of polymers of large chain fatty acids, or mixtures thereof.

[0132] In an embodiment, the hydrocolloid binding agent is a natural polymer derivative hydrogel. Preferably, the hydrocolloid binding agent is selected from the group of sodium carboxymethyl cellulose (CMC), carboxyethyl cellulose (CEC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), ethyl cellulose (EC), hydroxypropylmethyl cellulose (HPMC), hydroxyethylmethyl cellulose (HEMC), hydroxyethylpropyl cellulose (HEPC), bacterial cellulose (BC), cellulose nanofiber (CNF), cellulose nanocrystals (CNC), microfibrillated cellulose (MFC), hydroxypropyl methylcellulose phthalate, or mixtures thereof.

[0133] In an embodiment, the hydrocolloid binding agent is carboxymethyl cellulose or sodium carboxymethyl cellulose.

[0134] In an embodiment, the binding agent is present in a concentration between 0.1% wt. and 6% wt. of hydrogel before drying, preferably between 0.5% wt. and 5% wt., more preferably between 1% wt. and 4% wt.

[0135] In an embodiment, the gelling agent from the family of polysaccharide hydrocolloids in the rehydratable hydrogel of the composite layer (2) is selected from, but not limited to, sodium alginate, gellan gum, carrageenan, guar gum, xanthan gum, locust bean gum, gum arabic, pectin, or modified starch.

[0136] In an embodiment, the gelling agent is present in a concentration between 0.1% wt. and 6% wt. of hydrogel before drying, preferably between 0.5% wt. and 5% wt., more preferably between 1% wt. and 4% wt.

[0137] In an embodiment, the plasticizer in the rehydratable hydrogel of the composite layer (2) is selected from, but not limited to, polyvinyl alcohol (PVOH), ethylene glycol (EG), diethylene glycol (DEG), triethylene glycol (TEG), polyethylene glycol (PEG), propylene glycol (PG), glycerol, erythritol, sorbitol, mannitol, maltitol, xylitol, polyols, fatty acids, vegetal oils, or mixtures thereof.

[0138] In an embodiment, the plasticizer is present in a concentration between 1% wt. and 15% wt. of hydrogel before drying, preferably 2% wt. and 12% wt., more preferably 3% wt. and 8% wt.

[0139] In an embodiment, the hydrogel, may be formed through a chemical or physical cross-linking of individual polymer chains. The physical cross-linking can be achieved when the polymer is combined with ionic salts composed of an anion (di or trivalent) and a cation (di or trivalent). Chemical crosslinking can be achieved by the formation of intermolecular or intramolecular covalent bonds, where different cross-linkers can be used, which is understood by a person skilled in the art.

[0140] In an embodiment, the hydrogel formulation can be drop casted, coated, or wet laminated in the cellulose substrate layer (1) to produce the rehydratable hydrogel of the composite layer (2). The hydrogel formulation can be applied to the cellulose substrate layer (1) using several different coating techniques. Examples of these techniques include but are not limited to, rod, grooved rod, curtain,blade, slot-die, applicator roll, fountain, jet, short dwell, slotted die, bent blade, bevel blade, air knife, bar, gravure, size press, spray or any other suitable technique that allows the formation of a smooth coated surface.

[0141] In an embodiment, having the cellulose substrate layer with the hydrogel formulation provides a platform that allows the hydrogel to be polymerized outside / above the cellulose substrate layer.

[0142] In an embodiment, the composite layer comprises the rehydratable hydrogel and a suitable culture medium (2). In several embodiments, the culture medium may be impregnated in the rehydratable hydrogel.

[0143] In another embodiment, the culture medium is added to the cellulose-based microbiological culture device before rehydration of the hydrogel.

[0144] In an embodiment, any culture medium known in the field can be used in the composite layer (2) in an amount suitable to carry out microbial growth in the device.

[0145] In an embodiment, the composite layer (2) may further comprise other materials, such as a colorimetric agent, i.e. chromophores, or cell growth markers.Cellulose-based hydrophobic spacer (3):

[0146] In an embodiment, the cellulose substrate layer is coated with hydrophobic polymers to be used as a spacer to allow hydrogel swelling without compression.

[0147] In an embodiment, a fibrous substrate is a cellulose-based paper, wherein the cellulose fibers are raw and have no fillers (this substrate layer is made from the same or similar cellulosic material used and described in (1)). This substrate is layered on both sides with one or a mixture of hydrophobic cellulose derivative polymers. Said patterning composition is not detectable in the visible light spectrum, that is, under naked eye. The porous nature of the used substrates allows for this solution to coat homogeneously creating a continuous hydrophobic film.

[0148] In an embodiment, the hydrophobic polymer patterning composition comprise ethyl cellulose (EC). EC is a cellulose derivative that offers excellent film-forming properties; a solvent mix comprising at least one solvent selected from the group consisting of: alcohols (like methanol, ethanol, isopropyl alcohol, n-butanol, t-butanol, etc.); ketones (like acetone, butanone, isohexanone, etc.); esters (like ethyl acetate, isopropyl acetate, n-butyl acetate, etc.); N-Methyl-2-pyrrolidone (NMP); Propylene Glycol; Glycols (e.g., Ethylene Glycol); Glycerol: Glycerol, also known as glycerine; Diacetone Alcohol (DAA); Cellosolve Solvents (e.g., Ethylene Glycol Monoethyl Ether); Dipropylene Glycol (DPG); preferably, comprising ethanol and diacetone alcohol, wherein: the ethanol and the diacetone alcohol are provided in volume ratios ranging from 10:90 - 90:10 preferably 20:80, 30:70, 40:60, 70:30, 80:20; with a content of weight of a solid polymer (for ex. ethyl cellulose) from 1:1 up to 1:500, the mixture wasstirred at a temperature range within room temperature (around 20 degrees Celsius °C) and up to 70 °C).

[0149] In an embodiment, a hydrophobic polymer (3) is coating the cellulose substrate layer and is precisely shaped into the desired design, by adhesive assisted lamination onto the cellulose substrate (1).Polymeric encapsulation layer (4):

[0150] In an embodiment, the device contains at least one encapsulation layer (4) to allow for incubation at optimal bacterial growth temperatures with limited water evaporation, wherein the encapsulation can be flexible or non-flexible polymers allowing for a flexible stick-on surface device or a hard case encapsulation for high sample volume applications.

[0151] In an embodiment, this humidity barrier layer / encasing comprises watertight polymers, wherein said polymer composition is not detectable in the visible light spectrum (transparent polymer material to allow the visualization of the device of the present disclosure without color change / interaction). This ensures environmental protection, allows temperature-controlled incubation, and supports high-volume sample testing. These innovations enable the device's use in shipping, food packaging, or clinical settings.

[0152] In an embodiment this humidity barrier can be made out of several possible materials, such as: plastic lamination, varnish (any type and any application method), commercial PVA waterproof (any application method), any commercial barrier coatings, such as (and not limited to) bio-polymers (PLA); PE, PP, PET, EVOH and PE polymer structure, lignin-based coatings, polyvinyl alcohol (PVA), alkene ketene dimer (AKD), among others. In all the presented options the material can be used as a thin flexible layer laminated on top of the device; or used as bulk material and have a structured case able to hold higher sample volume and / or being able to be used as a standalone device. Both encapsulation types are introduced to allow the use of the present disclosure in innovative and distinct ways when compared to available technologies. The flexible thin encapsulation allows for a direct contact between the device and produce while the "hard shell" casing allow for the integration into containers for large volume liquid samples like the ones found in clinical setting (urine) or in the food and beverage industries.Antibiotic susceptibility testing / E-test

[0153] In an embodiment, the cellulose-based microbiological culture device comprises printed or deposited antibiotics on the rehydratable hydrogel of the composite layer for drug susceptibility testing and colorimetric detection as shown in Fig. 24. The incorporation of antibiotic gradient in the disclosedcellulose-based microbiological culture device is done by directly depositing or printing at least one antibiotic solution in the rehydratable hydrogel of the composite layer (2).

[0154] The present disclosure is innovative in concept since it is the first to integrate the antibiotic gradient, colorimetric detection, and culture media in one device as shown in Fig. 24. Working with an integrated culture media suppresses the need of a separate culture dish for this test, reducing waste material and analysis time, while providing a visual color-based direct result. The device is configured to provide a direct visual readout (e.g., blue-to-purple or pink-to-yellow colour shifts). A ruler like structure is arranged in the cellulose substrate layer to facilitate the scoring of samples. An antibiotic gradient is added to the rehydratable hydrogel of the composite layer in individualized cavities, namely testing zones or spots, and is aligned with the ruler like structure. These features enable quick and easily interpretable results by untrained users without requiring laboratory equipment.

[0155] In an embodiment, at least one antibiotic gradient is deposited on sequentially aligned cavities, namely testing zones or spots, previously infused with growth media. The step of applying a hydrogel formulation in the cellulose substrate layer (1) forming at least a layer zone comprising an antibiotic gradient may be carried out in such a way to form a pattern in specific regions of the rehydratable hydrogel of the composite layer (2).Method

[0156] The present disclosure also relates to a method of producing the cellulose-based microbiological culture device comprising the following steps:- impregnating a cross-linker agent in a cellulose substrate layer (1);- applying a hydrogel formulation in the cellulose substrate layer (1) forming at least one hydrogel layer (2);- drying the device at a temperature between 20 °C) to 50 °C;- washing the device in water for a period not bigger than 20 minutes;- drying the device at a temperature between 20 °C to 50 °C ;- Spot, individualization and removal of hydrogel in unnecessary areas (non-contact process);- adding a culture medium solution to the rehydratable hydrogel layer individualized cavities, namely testing zones or spots, and drying the device at a temperature between 20 °C to 50 °C; and - lamination of all the device layers.

[0157] In an embodiment, after applying the hydrogel formulation, the device can be washed with water for a period of less than 20 min and then dried at a temperature between 20 °C to 50 °C.

[0158] In an embodiment, the applying step is repeated according to the number of rehydratable hydrogel layers desired to form the composite layer (2).

[0159] In an embodiment, for devices of the present disclosure with more than one cellulose substrate layer, the method further comprises a step of stacking and bonding at least two cellulose substrate layers on top of each other.

[0160] In an embodiment, the method of the present disclosure further comprises the steps of depositing / printing at least one antibiotic gradient solution in the rehydratable hydrogel of the composite layer (2) comprising a colorimetric agent, to obtain an E-test like device.

[0161] In an embodiment, the hydrogel formulation comprises at least one binding agent from the family of cellulose-derived hydrocolloids, at least one gelling agent from the family of polysaccharide hydrocolloids and a plasticizer, as described above.

[0162] In an embodiment, the device is dried with hot air or infra-red lamps or any other suitable technique to dehydrate the hydrogel. Alternately to hot air drying, the hydrogel can also be lyophilized.

[0163] In an embodiment, the method according to the present disclosure also enables the cavities, namely testing zones or spots, individualization of the rehydratable hydrogel of the composite layer (2), as the hydrogel formulation can be deposited only at selected and defined regions of the cellulose substrate layer (1). This procedure allows localized antibiotic rich regions on the rehydratable hydrogel of the composite layer (2), allowing for antibiotic susceptibility testing. Moreover, the antibiotic can be deposited in controlled amounts at defined regions allowing the production of antibiotic gradients over the rehydratable hydrogel of the composite layer (2), hence creating a device that can determine the minimum inhibitory concentration (MIC) of microorganisms (such as E-test).

[0164] The embodiment of the present disclosure comprising an array of layer zones impregnated with an antibiotic can be combined with guide markers printed on the cellulose substrate layer (1) providing an easy score of susceptibility.

[0165] In an embodiment, once produced, the cellulose-based microbiological culture device can be encapsulated on an impermeable polymer material, allowing for standalone use.

[0166] The present disclosure further relates to the use of the cellulose-based microbiological culture device for the culture, detection, characterization, identification, and enumeration of microorganisms. This device is also suitable, but not limited, to be used with biological samples, for example urine, food and beverage samples, or environmental samples.

[0167] In an embodiment, the device of the present disclosure can be used for contact inoculation by contact with a surface.

[0168] Surface monitoring methods are used to evaluate the effectiveness of hygiene procedures and overall cleanliness of surfaces. Surface monitoring tests use contact plates or swabs according to international organization for standardization (ISO) standards. The swab method is suitable for unevensurfaces; while contact plates only allow for smooth surfaces. However, contact plates allow for consistent and quantitative contamination results. Additionally, swabs can cover larger areas when compared to contact plates. The present disclosure is suitable to be used both via contact inoculation and swab methods, allowing for a wide range of application scenarios without the use of extra disposable materials. The present disclosure can be used as a standard contact plate using standard protocols. When pressed on a presoaked test area, the device passively absorbs the liquid into the device activating the hydrogel cavities, namely testing zones or spots, collecting any viable microorganisms that may be present. Once analyzed, the level of growth is reported by the color change in each spot containing bacteria specific colorimetric and / or chromogenic growth media. Comparing the level of growth pre and post sanitization provides insight into the efficacy of sanitization or disinfection programs. Because not all surfaces are flat and / or smooth, surface swabbing can also be implemented. The fibrous cellulosic material and the hydrogel structure allows the present disclosure to be used to directly swab the surface without deformation or degradation of the growth media structure. Moreover, a controlled amount of water can be added to the test area allowing for the inoculation and hydration of the media to occur in one single step while increasing the efficiency of sample collection. This approach creates the advantage of a single test for multiple surface types and coverage areas in a single product.Usability and technical capabilities

[0169] In an embodiment, the device is placed under a camera apparatus for real-time color monitoring. The continuous analysis of color change in simple and / or multiple spot tags correlates with the initial bacterial contamination levels, allowing the use of the present disclosure to perform quantitative contamination analysis.

[0170] In an embodiment, the device is placed inside a large volume polymer container. This integration together with the hydration means side paper extension allows for the device to be in contact with the sample during the entire incubation process. This integration together with a multi spot (or cavity) device further introduces the capability to detect, identify and quantify multiple microorganisms in a large volume sample (>lmL) in a single device and incubation period while using a fraction of the growth media.

[0171] In an embodiment, the device is attached directly on the surface of food products allowing it to be inoculated with the water released during storage. The device self inoculates and allows for the proper hydrogel hydration, hence bacterial growth. If / when the food products are not kept at ideal storage temperatures / conditions, the device facilitates bacterial growth and colorimetric detection as such.

[0172] In an embodiment, the device is attached to the side of food product trays. The trays are designed to accumulate residual water onto a collection area where the device is secured. Device can be placed inside the tray or outside if the hydration means is extended to the inside to collect the sample.

[0173] In an embodiment, the device is placed inside a hard polymer low profile case to be directly inoculated with low volume samples or to be used in direct contact with food products, while reducing the risk of food contamination with the device components.Results

[0174] In an embodiment, Fig. 8 shows front, side, and back views of one of the proposed encapsulation embodiments of the device of the present disclosure. The opaque sheet covering the adhesive can be easily removed to allow the gluing of the device to a desired surface. The polymer prevents excessive evaporation to stimulate bacterial growth, while the inoculation strip is free to allow passive inoculation of liquid samples. Fig. 8 also displays the application of said encapsulation method, with devices glued onto the bottom and side surfaces of a typical household plastic container. This method grants the present disclosure great flexibility and ease of use, being able to be applied to a diverse range of surfaces, reducing the need for extra laboratory steps.

[0175] In an embodiment, Fig. 9 shows 3D-prototypes of other embodiments of the present disclosure, which comprise a hard-shell encapsulation for the device. In the embodiment shown on the left, the device can be inoculated via passive capillarity via the inoculation strip, while the embodiment on the right is for direct contact inoculation, via small holes in the bottom of the polymer capsule. This hardshell encapsulation grants better protection of the devices for certain applications.

[0176] In an embodiment, Fig. 10 shows a 3D printed prototype of one of the embodiments proposed in this document. It is comprised of a transparent polymer cup, with a chamber for sample deposition and a support for placement of the device of the present disclosure. The inoculation strip in the device enables passive fluidic transference of the liquid sample to the sensing composite layer comprising the hydrogel and the culture medium, which can be integrated with a chromogenic, fluorogenic or other type of culture media. The cup chamber can be made in different sizes, allowing for detection and / or identification of microorganisms in large sample volumes, which cannot be tested in conventional petri dishes.

[0177] In an embodiment, Fig. 11 shows the device of the present disclosure in a single spot (or cavity) embodiment, integrated with different culture media (see Table 1 for media list) after inoculation and 24-hour incubation with Enterococcus faecalis in liquid sample. This test shows the capability of the tags for selective detection of bacteria depending on the used culture media, as only some of the used media produced a color change when exposed to this bacterial species.

[0178] Table 1 - List of chromogenic media mixes used in Fig. 11 tests.

[0179] In an embodiment, Fig. 12 shows the device of the present disclosure in a single spot (or cavity) embodiment with different culture media incorporated in the hydrogel layer. Top row - RAPID Coliform ChromoSelect Broth (Merck); 2ndrow - Enterococci ChromoSelect Broth (Merck); 3rdrow - MacConkey Broth; bottom row - Mueller-Hinton Broth combined with Tetrazolium Blue Chloride indicator. The devices were inoculated with distinct microbial species and incubated 24-48 hours (from left to right: control - no bacteria, E. coli, E. faecalis; Salmonella spp. and Pseudomonas aeruginosa). This test further proves the capability of the present disclosure to be integrated with different culture media, which enables a selective identification of distinct species of microorganisms.

[0180] In an embodiment, the selectivity of microorganism detection is completely dependent on the type of culture media chosen, and not on the present disclosure, which serves as a way to preserve, and isolate said culture media.

[0181] In an embodiment, Fig. 13 shows the device of the present disclosure in the multi spot (or cavity) embodiment, with 4 different chromogenic media, isolated into hydrogel spots (or cavities). Top left - RAPID Coliform ChromoSelect Broth; top right - Enterococci ChromoSelect Broth; bottom left - VRBG (Violet Red Bile Glucose) Broth; bottom right - Half-Fraser Broth with Ammonium Ferric Citrate supplement. The devices were inoculated with distinct microbial species and incubated for 48 hours (bottom row). Results were also taken at 24 hours (top row) From left to right: control - no bacteria, E. coli, E. faecalis; Salmonella spp. and Pseudomonas aeruginosa and Listeria spp.). This test evidences the capacity of the present disclosure for multiplexed analysis of distinct bacterial species using a single device, without cross-contamination of isolated spots (or cavities).

[0182] In an embodiment, Fig. 14 shows the present disclosure being used to detect bacterial growth in spoiling fish (sole). Some devices were placed directly on top of fish, and others were glued to the inside wall of the polystyrene box where the fish is stored, in order to absorb the wastewater that results from the melting of the ice and spoiling of the fish. This test demonstrates the flexibility and ease of use ofthe device of the present disclosure, easily placed in different locations, even directly on top of samples, to test for microbial growth.

[0183] In an embodiment, Fig. 15 shows a close-up of the device of the present disclosure, in the single-spot (or cavity) embodiment, used for detection of bacterial growth in a test with spoiling fish. In the left columns, devices contain different culture media incorporated in the hydrogel layer (RAPID - RAPID Coliform ChromoSelect Broth; EC - Enterococci ChromoSelect Broth; VRBG - Violet Red Bile Glucose Broth; MC - MacConkey Broth) with the devices being placed directly on top of the spoiling fish, with the bottom absorbing layer (i.e., cellulose substrate) contacting the skin of the fish. In the right column, devices contain the same culture media (EC - Enterococci ChromoSelect Broth) but are placed not only directly on top of fish, but also glued to different locations in the box where the fish is stored, with inoculation being performed by capillarity, via the absorbing bottom layer strip (cellulose substrate). This further demonstrates the capabilities of the present disclosure to be integrated with distinct culture media and to be used in varied locations, granting it a tremendous flexibility and adaptability to unique cases where conventional plating is not adequate.

[0184] In an embodiment, Fig. 16 shows a close-up of the present disclosure, in a single spot (or cavity) embodiment, in spoiling fish tests. The devices were integrated with different culture media in the sensing hydrogel layer, with pictures taken during several days, as the fish spoiled.

[0185] In an embodiment, Fig. 17 shows the peaks of bacterial growth during the test, obtained by RGB analysis of the devices over the course of several days and algorithmic analysis. The blue area indicates the time at which ice was present in the box and the red area signals the time at which there is clear olfactory detection of rotting smell. This test demonstrates how the present disclosure can be used to detect growth spurts of distinct bacterial species in samples where conventional plating is not applicable.

[0186] In an embodiment, Fig. 18 shows the graphs of bacterial growth obtained by RGB analysis of the devices in spoiling fish tests, combining all the curves from each different culture media, in different placements. The blue area indicates the time at which ice was present in the box and the red area signals the time at which there is clear olfactory detection of rotting smell. This graph demonstrates how the present disclosure can be used to signal important peaks of bacterial growth in fish samples, and thus serve as a "timeclock" indicator of fish spoilage, possibly enabling users to preemptively detect inadequate storage conditions before complete degradation of the product.

[0187] In an embodiment, Fig. 19 shows the present disclosure in a single spot (or cavity) embodiment, in another spoiling fish test, separate from the one presented in previous figures. The hydrogel was integrated with different culture media, namely RAPID Coliform ChromoSelect Broth (RAPID), Enterococci ChromoSelect Broth (EC), Half-Fraser Broth with Ammonium Ferric Citrate supplement (HF+AI), Mueller-Hinton Broth with Tetrazolium Blue Chloride indicator (MH+IR), MacConkey Broth (MC)and Violet Red Bile Glucose Broth (VRBG) and placed directly on top of fish and glued to the inside wall in the polystyrene container.

[0188] In an embodiment, in the same test as Fig. 19, Fig. 20 shows devices in the multi-spot (or cavity) embodiment, with 3 alternate combinations of the culture media referenced above, during different days of the experiment. These results further reinforce the capability of the present disclosure to be used for applications where conventional Petri dish-based plating is inadequate, the capability of the devices to be used with different types of culture media and the feasibility of multiplexed analysis using only one device.

[0189] In an embodiment, Fig. 21 shows the present disclosure, in a single spot (or cavity) embodiment, with the hydrogel layer integrated with Enterococci ChromoSelect Broth and exposed to Enterococcus faecalis at different inoculum dilution factors namely, from left to right: 0; 10'6; 10'5; 10'4; 10'3. The devices were left to incubate for 18 hours at 36 °C and pictures were taken every hour to observe evolution of color change.

[0190] In an embodiment, Fig. 22 shows the graph obtained by plotting the color intensity of the devices with time, at the different dilution factors, obtained by RGB analysis of the devices.In an embodiment, Fig. 23 shows the graph obtained by plotting the slope of the curves with the dilution factor of the devices. The slope was obtained from the trendline of the growth phase in each curve from the color / time graph of Fig. 22. This test evidence how the present disclosure is used for a quantitative analysis of bacterial load, by combination with an automatic detection system which takes pictures at specific times and analyses the change in the color of the spots (or cavities). Then via a specific algorithm, it identifies the slope of the growth phase of color change and matches it to a specific bacterial load.

[0191] In an embodiment, Fig. 24 shows the present disclosure in an E-test-like embodiment, after inoculation and 24h growth of a liquid sample containing Escherichia Coli. Chromogenic culture media for identification of coliform bacteria (RAPID Coliform ChromoSelect Broth) and an antibiotic gradient (rising concentration from left to right) were deposited onto the hydrogel layer to create a composite layer (2). These results evidence the capability of the present disclosure to be used for the determination of the MIC of microorganisms.

[0192] An aspect of the present disclosure further relates to the applications of the device, which include, without limitation, food safety testing, water quality monitoring, and clinical assays.

[0193] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0194] The terms "determining / ' "measuring / ' "evaluating / ' "assessing / ' "assaying / ' and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0195] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0196] Notably, the figures and examples above are not meant to limit the scope of the present disclosure to a single implementation, as other implementations are possible by way of interchange of some or all the described or illustrated elements. Moreover, where certain elements of the present disclosure can be partially or fully implemented using known components, only those portions of such known components that are necessary for an understanding of the present disclosure are described, and detailed descriptions of other portions of such known components are omitted so as not to obscure the disclosure. In the present specification, an implementation showing a singular component should not necessarily be limited to other implementations including a plurality of the same component, and vice-versa, unless explicitly stated otherwise herein. Moreover, applicants do not intend for any term in the specification or claims to be ascribed an uncommon or special meaning unless explicitly set forth as such. Further, the present disclosure encompasses present and future known equivalents to the known components referred to herein by way of illustration.

[0197] The foregoing description of the specific implementations will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the relevant art(s), readily modify and / or adapt for various applications such specific implementations, without undue experimentation, without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed implementations, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpretedby the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one skilled in the relevant art(s). It is to be understood that dimensions discussed or shown are drawings accordingly to one example and other dimensions can be used without departing from the disclosure.

[0198] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes can be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the invention encompassed by the present disclosure, which is defined by the set of recitations in the following claims and by structures and functions or steps which are equivalent to these recitations.

[0199] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0200] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0201] The following dependent claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. Device for detecting a microorganism in a sample, comprising: a cellulose substrate with a plurality of cavities; a composite layer within each cavity, comprising a rehydratable hydrogel and a culture medium for cultivating a microorganism; wherein the cellulose substrate is configured to absorb the sample via capillary action into the bottom of the cavities, to facilitate interaction between the sample and the composite layer for the detection of a microorganism in the sample.

2. Device for detecting a microorganism in a sample, comprising: a cellulose substrate; a composite layer on the top of the cellulose substrate, wherein said composite layer comprises a rehydratable hydrogel and a culture medium for cultivating a microorganism in the top of the cellulose substrate; wherein the cellulose substrate is configured to absorb the sample via capillary action into the bottom of the cavities, to facilitate interaction between the sample and the composite layer for the detection of a microorganism in the sample; wherein the cellulose substrate surrounding the composite layer comprises at least one hydrophobic compound or a mixture of such compounds to prevent the spread of the sample and culture medium outside the composite layer.

3. Device according to previous claim 1, wherein the cellulose substrate surrounding the composite layer comprises at least one hydrophobic compound or a mixture of such compounds to prevent the spread of the sample and culture medium outside the composite layer.

4. Device according to any of the previous claims, wherein the top layer is encapsulated by an involucre wherein said involucre comprises a water impermeable polymer.

5. Device according to any of the previous claims, wherein the cellulose substrate is a cellulose fibrous layer.

6. Device according to any of the previous claims, wherein the cellulose substrate comprises a plurality of layers.

7. Device according to the previous claim, wherein the plurality of layers of the cellulose substrate ranges from 2-10 layers; preferably 2-5 layers, more preferably 3-5.

8. Device according to any of the previous claims, wherein the cellulose substrate is paper.

9. Device according to any of the previous claims, wherein the cellulose substrate is selected from the group consisting of: bleached softwood fibers, unbleached softwood fibers, bleached hardwood fibers, unbleached hardwood fibers, cotton fibers, or mixtures thereof.

10. Device according to any of the previous claims, wherein the cellulose substrate grammage ranges from 50 g / m2- 500 g / m2; preferably from 100 g / m2- 400 g / m2; more preferably from 200 g / m2- 300 g / m2.

11. Device according to any of the previous claims, wherein the thickness of the cellulose substrate ranges from 300 pm - 1 mm, preferably 350 pm - 600 pm; more preferably 400 pm - 500 pm.

12. Device according to any of the previous claims, wherein the cellulose substrate further comprises at least one cross-linker agent and / or at least one wet strength agent.

13. Device according to any of the previous claims, wherein the composite layer is impregnated or coated.

14. Device according to any of the previous claims, wherein the composite layer comprises a plurality of sub-layers.

15. Device according to any of the previous claims, wherein the composite layer comprises at least 1 to 4 sub-layers.

16. Device according to any of the previous claims, wherein the composite layer is arranged on top of or partially integrated in the cellulose substrate.

17. Device according to the previous claim, wherein the composite layer is partially integrated in the cellulose substrate, forming an interface layer.

18. Device according to the previous claim, wherein the interface layer has a thickness between 0.1 pm - 500 pm; preferably 1 pm - 100 pm, more preferably 5 pm - 50 pm.

19. Device according to any of the previous claims, wherein the composite layer thickness ranges from 5 pm - 500 pm; preferably 10 pm - 300 pm, more preferably 20 pm - 200 pm.

20. Device according to any of the previous claims, comprising a plurality of perforations in at least one of the layers or sublayers of the substrate and / or the composite layer.

21. Device according to any of the previous claims, wherein the hydrogel grammage in the substrate ranges from 20 g / m2- 600 g / m2; preferably 50 g / m2- 400 g / m2, more preferably 100 g / m2- 300 g / m2.

22. Device according to any of the previous claims, wherein the amount of culture medium in the composite layer ranges from 5 g / m2- 500 g / m2; preferably 20 g / m2- 400 g / m2, more preferably 30 g / m2- 300 g / m2.

23. Device according to any of the previous claims, wherein the composite layer comprises at least one binding agent, one gelling agent, and one plasticizer, or mixtures thereof.

24. Device according to any of the previous claims, wherein the culture medium of the composite layer in each cavity comprises variations in the composition or different concentrations to enhance specificity for the detection of different microorganisms.

25. Device according to any of the previous claims, wherein in the composite layer further comprises antimicrobial agent; preferably an antibiotic.

26. Device according to any of the previous claims, wherein the amount of antimicrobial agent in each cavity is different.

27. Device according to any of the previous claims, wherein the cavity, preferably a plurality of cavities, in the cellulose substrate layer has a gradient of antimicrobial agent concentration.

28. Device according to any of the previous claims, wherein the composite layer further comprises a dye and / or chromogenic agent.

29. Device according to any of the previous claims, wherein the composite layer further comprises at least one cell growth marker.

30. Device according to any of the previous claims, wherein the hydrophobic compound further comprises at least one transparent and hydrophobic polymer.

31. Device according to any of the previous claims, wherein the hydrophobic compound is selected from the group consisting of: ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose oleate, cellulose laurate, silylated cellulose, or cellulose acetate phthalate, or mixtures thereof.

32. Device according to the previous claim 4, wherein the encapsulation involucre further comprises a polymer.

33. Device according to any of the previous claims, wherein the polymer is a flexible polymer and / or a hard polymer.

34. Device according to claim 33, wherein the flexible polymer is selected from a list consisting of: polyethylene, polypropylene, polyvinyl chloride, polyurethane, silicone rubber, thermoplastic elastomers, polydimethylsiloxane, polycarbonate, nylon, polyamide, ethylene vinyl acetate, polyvinylidene fluoride, ethylene propylene diene monomer, natural rubber, styrene-butadiene rubber, or polyisoprene, or mixtures thereof.

35. Device according to claim 33, wherein the hard polymer is selected from a list consisting of: polytetrafluoroethylene, polyvinyl chloride, polymethyl methacrylate, polystyrene, polycarbonate, polyethylene terephthalate, polypropylene, acrylonitrile butadiene styrene, polyetheretherketone, polyoxymethylene, high-density polyethylene, low-density polyethylene, polyphenylene sulfide, polyvinylidene fluoride, polyamide, or nylon, or mixtures thereof.

36. Device according to any of the previous claims, further comprising a handling zone for the transportation or placement of the device.

37. Device according to the previous claim, wherein the handling zone is connected to rehydration means.

38. Kit for detecting a microorganism in a sample, comprising the device described in any of the previous claims, preferably wherein said kit further comprise a sample container.

39. Method for producing the device for detecting a microorganism in a sample, comprising the steps of:impregnating a cross-linker agent in the cellulose substrate; applying a rehydratable hydrogel in the cellulose substrate according to the number of rehydratable hydrogel layers desired, forming at least one hydrogel layer; drying the device at a temperature between 20 - 50 °C; washing the device in water for a period inferior to 20 minutes; adding one or several culture media solutions to each testing cavities; drying the device at a temperature between 20 - 50 °C; individualization of the cavities and removal of hydrogel in unnecessary areas; adding a culture medium solution to the rehydratable hydrogel individualized cavities; drying the device at a temperature between 20 - 50 °C; and, lamination of all the device layers.

40. Method for producing the device for detecting a microorganism in a sample according to claim 39, further comprising depositing at least one antibiotic gradient solution and a colorimetric agent in the composite layer.

41. Method for producing the device for detecting a microorganism in a sample according to claims 39 to 40, further comprising the lyophilization of the hydrogel as the last step.

42. Use of the device according to any of the previous claims, for the culture, detection, characterization, identification, and enumeration of microorganisms.

43. Use of the device according to the previous claim, comprising the use with biological, food, beverage or environmental samples.

44. Use of the device according to claims 42 to 43, comprising contact inoculation by direct contact with a surface.

45. Use of the device according to claims 42 to 44, comprising inoculation via passive capillarity using an inoculation strip.

46. Method for detecting a microorganism in a sample, comprising the use of the device according any of the claims 1- 37 comprising the step of contact the sample with the bottom of the device.

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