Microfluidic device for liquid sample processing, methods of production and uses thereof

Abstract

The present disclosure relates to a microfluidic device for liquid sample processing comprising an inlet, an outlet, a chamber, a feeding channel and a collection channel, fluidically connected, wherein the inlet, feeding channel, chamber, and collection channel are arranged at successive vertical levels such that the chamber is positioned below the inlet and the collection channel is positioned below the chamber; and wherein the chamber comprises a fibrous polymeric substrate. The use of the microfluidic device as cell culture platform and / or drug screening platform, as a filtration and / or isolation platform, in particular filtration and / or isolation of cells, microplastics, pollutants, or microparticles in a liquid sample and / or also as an in vitro diagnosis platform are also disclosed. The methods to obtain the microfluidic chip and its operating workflow are also disclosed.

Description

D E S C R I P T I O NMICROFLUIDIC DEVICE FOR LIQUID SAMPLE PROCESSI NG, METHODS OF PRODUCTION AN D USES THEREOFTECHN ICAL FIELD

[0001] The present disclosure relates to the field of microfluidics and their multifaceted applications in biomedical diagnostics, material sciences, and environmental monitoring. The disclosure encompasses methods of production of the microfluidic device and its diverse uses across multiple disciplines.BACKG ROU N D

[0002] The development of methods for the isolation of microparticles, notably microplastics, from environmental samples has gained significant momentum due to increasing environmental concerns. Microplastics are widely recognized as persistent environmental contaminants [1], Large amounts of plastic waste have been discovered in the oceans, which degrade through processes such as abrasion, leading to microplastics that can be ingested by marine organisms, eventually entering the human food chain. This poses a potential risk to human health. Additionally, microplastics can act as carriers for numerous bacteria and organic compounds, further increasing the hazard to both ecosystems and human health [2], The isolation and extraction of microplastics from simple matrices, such as drinking water samples and other beverages, have been performed using various methods. These methods provide rapid but preliminary results due to certain methodological limitations. Basic isolation techniques often involve filtering clean water samples, where samples are passed through filter papers or meshes for visual inspection and chemical verification [3], However, different laboratories employ different filters and pore sizes, leading to inconsistency in data. The standardization of isolation methods is critical to producing reproducible and comparable data worldwide.

[0003] In addition to microplastics, other non-biological entities such as industrial microparticles and airborne pollutants are also of great concern, particularly in environmental monitoring and pollution control. Industrial microparticles, including heavy metals and particulate matter from manufacturing processes, can contaminate water, soil, and air. These pollutants are not only hazardous to the environment but can also contribute to respiratory diseases and other health conditions when inhaled or ingested. The ability to isolate, quantify, and analyze these non-biological entities is crucial for assessing their impact on public health and environmental safety. Conventional methods for detecting these microparticles often involve filtration or centrifugation, but such methods lack sensitivity and precision, especially when dealing with heterogeneous samples.

[0004] In the biomedical field, the detection and monitoring of disease-related biomarkers, particularly circulating tumor markers (CTMs), play a crucial role in the early diagnosis, prognosis, and treatment of various malignancies [4, 5], CTMs, such as circulating tumor cells (CTCs) and circulating extracellular vesicles (cEVs), including exosomes, have emerged as significant biomarkers due to their ability to provide real-time insights into tumor dynamics and metastasis [6], However, isolating and analyzing these biomarkers is challenging due to their low abundance in body fluids and the complex environment from which they must be extracted [7-9], In addition to cancer-related biomarkers, the detection of biomarkers for other diseases, such as cardiovascular diseases, neurodegenerative disorders, and autoimmune diseases, is of growing interest in biomedical diagnostics. Biomarkers such as cardiac troponins, amyloidbeta, and autoantibodies provide critical information for diagnosing and monitoring conditions like heart disease, Alzheimer's disease, and rheumatoid arthritis, respectively

[0010] , Similar to CTMs, the low abundance of these biomarkers in blood and other bodily fluids poses a significant challenge to conventional diagnostic methods, which often require large sample volumes, complex preparation steps, and extended processing times.

[0005] Conventional methods for capturing and analyzing CTMs and other biomarkers often rely on bulk processes that lack the sensitivity and specificity required for detecting rare events like the presence of CTCs or circulating DNA / RNA in the early stages of cancer or other diseases

[0011] , These methods generally require extensive sample preparation, including multiple enrichment and purification steps, making them time-consuming and less practical for rapid diagnostics in clinical settings [9],

[0006] Microfluidic technology has potential for addressing these challenges. Microfluidic devices, which can process small sample volumes with high precision and efficiency, have shown great promise in improving the detection and isolation of both biological and non-biological entities [12, 13], These devices have been recognized for their ability to handle heterogeneous samples, reduce sample preparation time, and provide high sensitivity in the detection of rare biomarkers such as CTMs, non-cancer-related biomarkers, and even environmental microparticles [14, 15], Despite these advancements, there is a continued need for more versatile, cost-effective, and practical devices that can address both biomedical and environmental applications.

[0007] Document US9606086 B2 describes a microfluidic device for separating suspended particles and cells using a tilted surface acoustic wave method. However, this device comprises a complex setup since it requires a surface acoustic wave (SAW) generator, making it challenging for widespread use in clinical and environmental settings.

[0008] Document US10821439 B2 describes a microfluidic apparatus comprising one or more sequestration pens designed to isolate micro-objects such as microparticles and cells by changing the orientation of the microfluidic apparatus with respect to a globally active force, such as gravity. However,this device requires extended time to isolate microparticles, limiting its practicality for high-throughput applications.

[0009] Document WO 2016 / 019401 Al describes microfluidic devices and methods for the enrichment and detection of rare cells such as CTCs and other biomarkers, including proteins and DNA / RNA from biological fluid samples. However, in this device, cells are captured in a microfluidic chamber coated with a cell capture ligand, which complicates cell recovery and downstream analysis.

[0010] Document CN 109852530 B describes microfluidic devices that use channels of varying heights to capture, lyse, and detect the nucleic acids of CTCs. However, this device involves cell lysis and does not allow for intact cell recovery, thus limiting its utility in applications that require viable cells.

[0011] Document WO 2012 / 138882 A2 describes microfluidic chips for cell detection from a fluidic source. However, in this device, cells are bound to magnetic beads for detection, and the removal of the beads requires an additional step, complicating the process.

[0012] Document US 11262361 B2 describes a microfluidic device for the capture of rare cells. However, this device employs antibody-functionalized thin-film photodegradable hydrogels as the capture surface for rare cells, a procedure that can be expensive, limiting its feasibility for routine clinical or environmental use.

[0013] Document US 11808767 B2 describes a microfluidic device for detecting, separating, and analyzing cells in a fluid sample. However, the device uses antibodies for cell detection, and it does not support the recovery of intact cells.

[0014] These facts are disclosed to illustrate the technical problem addressed by the present disclosure.GEN ERAL DESCRIPTION

[0015] The present disclosure relates to a microfluidic device designed for the efficient capture and isolation of microparticles from liquid samples, namely biological and non-biological liquid samples. This versatile and efficient microfluidic device is capable of isolating and analyzing both biological and non- biological entities, including CTMs, other disease-related biomarkers, and environmental microparticles, in a manner that is both cost-effective and practical for clinical and environmental applications.

[0016] Specifically, it introduces an innovative microfluidic device that integrates electrospun fiber nets (EFNs) within its architecture, enabling the selective capture and isolation of either biological or non- biological entities from liquid samples. The device effectively targets biological entities, such as CTMs, but also microparticles, pollutants, and contaminants found in various human, chemical and environmental samples.

[0017] In an embodiment, in the context of environmental applications, the microfluidic device is capable of isolating and analyzing a wide range of non-biological entities, including but not limited microspheres with diameters ranging from 8 to 20 pm, in particular 8, 10, and 20 pm; microplastics with diameters ranging from 10 to 140 pm, in particular mixed microplastic suspensions of 10-30 pm, 40-60 pm, and 100-140 pm; along with heavy metals, and other pollutants in air, water, and soil samples. This functionality is significant for environmental monitoring, contaminants and pollution assessment, and compliance with environmental regulations, providing crucial data for ecosystem health evaluation.

[0018] In another embodiment, for biomedical applications, the microfluidic device offers advanced capabilities in the isolation of biological entities such as CTCs and extracellular vesicles (EVs), which are critical for the early detection, diagnosis, and monitoring of various cancers and diseases. The device facilitates the capture, in vitro culture, and analysis of CTMs, thus contributing to the advancement of personalized medicine and targeted therapies.

[0019] The present disclosure also encompasses methods for quantifying both non-biological microparticles and biological markers, enhancing the accuracy and efficiency of diagnostic procedures, environmental assessments, quality control processes, and research applications. Thus, the disclosed microfluidic device can be applied in various fields, including but not limited to environmental monitoring, namely in the detection, isolation, and analysis of microparticles, pollutants, and pathogens in environmental samples for pollution assessment, water quality testing, and environmental research; material sciences, namely in particle sorting, formulation, and characterization in the development of new materials and products; biomedical diagnostics, such as in the early detection and monitoring of a wide range of cancers and diseases via biomarkers, aiding in timely intervention and improving patient outcomes; pharmaceutical development, namely in drug discovery and screening through the isolation, in vitro culture and analysis of cellular and extracellular components.

[0020] In additional embodiments, the microfluidic device serves as a versatile tool in environmental science for monitoring ecosystem health, detecting environmental hazards, and supporting conservation efforts. In biomedical fields, it aids in disease detection, diagnosis and classification, monitoring disease progression, conducting liquid biopsies, and facilitating personalized therapeutic strategies.

[0021] In an embodiment, the microfluidic device comprises a polymeric substrate with pore sizes ranging from 0.6 nm to 28.6 pm and porosities from 2% up to 59.9%, enabling the selective capture of target entities. This capability ensures the efficient and effective isolation of either biological or non- biological particles, aligning with the diverse applications of the microfluidic device.

[0022] In an embodiment, the disclosed microfluidic device integrates state-of-the-art electrospinning technology, which produces EFNs, with advanced microfluidic architecture to create a platform capable of capturing microparticles, CTMs, and other disease-related biomarkers. Surprisingly, the disclosedmicrofluidic device can efficiently capture particles spanning several orders of magnitude in size, from nanoscale vesicles ("'20-150 nm) to circulating cells (10-30 pm) and large non-biological microparticles and microplastics (10-140 pm), far exceeding the size range typically achievable with conventional microfluidic filters. In an embodiment, the disclosed microfluidic device allows for the efficient capture, isolation and in vitro culture of CTMs, namely CTCs, and other biomarkers obtained from patient samples.

[0023] Surprisingly, significant improvements over existing diagnostic platforms are achieved by the disclosed microfluidic device which combines the structural properties of biocompatible electrospun nanofibers with three-dimensional microfluidics. This integration enables the isolation and further culture of cells, namely CTMs, and other biomarkers in a minimally invasive manner. The vertical organization of the disclosed microfluidic device reduces cell damage and minimizes the distress by lowering hydrodynamic pressure, enabling in vitro cell culture afterwards.

[0024] In an embodiment, the disclosed microfluidic device comprises a biofunctional polymeric substrate, in particular a fibrous polymeric substrate (EFNs), which allows for the optimization of fiber diameter, pore size, and surface chemistry to enhance the capture efficiency of CTMs, including CTCs and circulating extracellular vesicles (cEVs), as well as other disease-related biomarkers. The EFNs within the microfluidic device play a pivotal role in improving the efficiency of biomarker capture by leveraging the physical properties of the fibers to isolate target entities based on size and other physicochemical properties. In an embodiment, the polymeric substrate is capable of selectively capturing CTMs and other biomarkers naturally present in body fluids, particularly blood plasma. CTMs and biomarkers isolated onto the EFNs can be maintained for longer periods or in higher concentrations, thus facilitating downstream analysis.

[0025] In an embodiment, the polymeric substrate comprises a polymer selected from polycaprolactone (PCL), polymethyl methacrylate (PM MA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyethylene oxide (PEO), polyglycolic acid (PGA), collagen, gelatin, chitosan, cellulose, cyclic olefin copolymer (COC), or mixtures thereof. In a preferred embodiment, the polymeric substrate comprises PCL and PMMA. The selection of the polymer material allows for tuning the mechanical properties, biodegradability, and surface chemistry of the EFNs to suit specific applications and target entities.

[0026] The present disclosure further refers to the method for producing the microfluidic device, which is designed with four bonded layers. In a top view, the chip features an inlet on the left, an outlet on the right, and a central circular chamber that houses the EFNs. In an embodiment, the method for producing the disclosed microfluidic device for liquid sample processing, in particular the capture of biological and non-biological entities, comprises: (i) the use of UV-photolithography to fabricate master molds containing the microfluidic channels; (ii) replica molding of the master mold structures using polydimethylsiloxane (PDMS); (iii) activation of the surfaces through oxygen plasma treatment using alow-pressure plasma system; (v) integration of the EFNs within the microfluidic chamber; (vi) characterization of the process in terms of microstructural fidelity and capture efficiency of biological and non-biological entities.

[0027] The application of the disclosed microfluidic device in in vitro diagnostic procedures and environmental monitoring is also disclosed. These devices are particularly suited for use in cancer diagnostics and the detection of other diseases, offering a non-invasive, sensitive, and specific approach to detecting and monitoring various malignancies and pathological conditions.

[0028] In an embodiment, the device of the present disclosure can be used in the early detection, monitoring and treatment of a wide range of cancers, including lung, breast, prostate, colorectal, gastric, liver, pancreatic, esophageal, bladder, non-Hodgkin lymphoma, leukemia, cervical, skin (melanoma and non-melanoma), ovarian, and kidney cancers, as well as other malignancies. Additionally, it can be applied to detect biomarkers associated with cardiovascular diseases, neurodegenerative disorders, and infectious diseases.

[0029] In another embodiment, the disclosed microfluidic device can be used on environmental applications, such as the detection and analysis of microplastics, pollutants, and other non-biological microparticles in water, air, soil, and biological samples. In an embodiment, the device can process biological matrices including saliva, semen, blood, breast milk, and other bodily fluids for the isolation and analysis of microplastics and contaminants. Furthermore, the disclosed device can be utilized in tissue engineering applications, organ or tissue disease models, drug discovery, drug screening, or as a tissue implant. The device is adaptable for various clinical and environmental settings, offering a significant advancement in diagnostics, personalized medicine, and environmental monitoring.

[0030] The present disclosure relates to a microfluidic device integrated with a fibrous polymeric substrate designed for the selective isolation of either biological or non-biological entities from liquid samples. The polymeric substrate exhibits a wide range of pore sizes from 0.6 nm to 28.6 pm and a porosity ranging from 2% up to 59.9%, optimizing the capture of various target entities. Specifically, the substrate is engineered to capture biological entities such as CTCs and circulating extracellular vesicles (cEVs) within a size range of ~20 nm to 30 pm. In contrast, the substrate can also accommodate non- biological entities, including microplastics and environmental pollutants, ranging from 10 pm to 140 pm.

[0031] In an embodiment, the microfluidic device incorporates an inlet for liquid sample entry, an intermediate chamber housing the polymeric substrate, and microfluidic channels designed to enhance fluid interaction for efficient entity capture. Methods for producing the device include UV- photolithography for mold creation and the integration of the polymeric substrate into the microfluidic structure. The device is applicable in diverse fields, including environmental monitoring, biomedicaldiagnostics, material sciences, and pharmaceutical development, thereby offering a versatile solution for effective isolation and analysis of either biological or non-biological particles in various samples.

[0032] The present disclosure relates to a microfluidic device comprising a polymeric substrate product for isolating biological and non-biological entities from liquid samples, wherein said microfluidic device comprises a fibrous polymeric substrate having pore sizes varying from 0.6 nm to 28.6 pm and a porosity ranging from 2% to 59.9%.

[0033] In an embodiment, the fibrous substrate is a woven or nonwoven mesh composed of synthetic polymers selected from polycaprolactone (PCL), polymethyl methacrylate (PMMA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyethylene oxide (PEO), polyglycolic acid (PGA), or natural polymers selected from collagen, gelatin, chitosan, cellulose, cyclic olefin copolymer (COC), or mixtures thereof.

[0034] In an embodiment, the electrospinning parameters are adjusted to control: Fiber diameter in the range of 100 nm to 2 pm; Pore size optimized for capturing biological entities within the range of ~20 nm to 30 pm, preferably 30 nm to 30 pm, specifically targeting circulating tumor cells (CTCs) [10 pm to 25 pm] and circulating extracellular vesicles (cEVs) [30 nm to 1 pm]. The pore architecture and three-dimensional fibrous network also facilitates the capture of non-biological entities across a broad size spectrum, from nanoscale particles (~20 nm) to large microplastics (10-140 pm), preferably from 20 nm to 50 pm, as well as environmental pollutants and microparticles ranging from 20 nm (e.g., certain nanoparticles) to several tens of micrometers (e.g., PM2.5 and larger particulate matter); Surface chemistry to facilitate biofunctionalization with antibodies or ligands.

[0035] In an embodiment, the substrate is biofunctionalized with specific antibodies or ligands to enhance the selective capture of target biological entities, including CTMs as circulating extracellular vesicles (cEVs).

[0036] In an embodiment, polymeric substrate product is for use in isolating non-biological entities such as microparticles, microplastics, pollutants, and contaminants from environmental samples including air, water, and soil.

[0037] It is also disclosed a diagnostic microfluidic device and analytical kit comprising: a polymeric substrate product and a microfluidic device configured to integrate the polymeric substrate for the capture and isolation of biological and non-biological entities from liquid samples.

[0038] In an embodiment, the microfluidic device comprises an inlet for liquid sample entry; an intermediate chamber housing the polymeric substrate product; microfluidic channels connecting the inlet to the intermediate chamber and the intermediate chamber to an outlet; an outlet to allow the processed sample to exit the microfluidic device;wherein the microfluidic channels are designed to optimize fluid interaction with the polymeric substrate for efficient capture of biological and non-biological entities.

[0039] In an embodiment, the length of the device is from 5 mm to 150 mm, preferably from 10 mm to 75 mm, more preferably about 35-42 mm. The inlet and the outlet each comprise a circular well having a width (diameter) from 200 pm to 10 mm, preferably from 500 pm to 5 mm, more preferably about 1 mm. The inlet well has a depth of about 3 mm and the outlet well has a depth of about 5 mm from the top surface of the device. The intermediate chamber has a diameter from 1 mm to 15 mm, preferably from 2 mm to 10 mm, more preferably about 6.5 mm. The microfluidic channels have a width from 10 pm to 2 mm, preferably from 100 pm to 1 mm, more preferably about 200 pm, and a height of about 250 pm.

[0040] It is also disclosed a method for producing the microfluidic device, comprising the following steps:(i) Producing an SU-8 master mold using UV-photolithography to define the microfluidic channel network;(ii) Casting a polydimethylsiloxane (PDMS) solution at a 10:1 ratio of pre-polymer to crosslinker onto the SU-8 master mold; (iii) Curing the PDMS for at least 30 minutes, preferably from 30 minutes to 1 hour, at about 70 °C to form PDMS replica moldings; (iv) Integrating the polymeric substrate into the intermediate chamber of the microfluidic device, which is designed to enhance the selective capture of biological entities, such as circulating tumor cells (CTCs) and extracellular vesicles (cEVs), as well as non-biological entities, including microplastics and environmental pollutants; (v) Bonding the PDMS layers to assemble the microfluidic device.

[0041] It is also disclosed a method of using the microfluidic device for the isolation and analysis of biological and non-biological entities from liquid samples, comprising: Introducing a liquid sample into the microfluidic device via the inlet; Allowing the sample to flow through the microfluidic channels and interact with the polymeric substrate in the intermediate chamber; Capturing target entities onto the polymeric substrate; Collecting the processed sample from the outlet; Analyzing the captured entities for diagnostic or environmental assessment purposes; Keeping viable CTCs in culture for further investigation.

[0042] In an embodiment, the biological entities include circulating tumor markers (CTMs), circulating tumor cells (CTCs), circulating extracellular vesicles (cEVs), including exosomes, microvesicles and apoptotic bodies, circulating nucleic acids such as circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cell-free RNA (cfRNA), microRNA (miRNA), long non-coding RNA (IncRNA), messenger RNA (mRNA), and extracellular RNA (exRNA). The biological entities further include proteins and peptides such as circulating enzymes, cytokines, chemokines, growth factors, and disease-associated biomarkers; viral particles, viral nucleic acids, and bacteriophages; bacteria, fungi, protozoa and their fragments; as well as other pathogenic or disease-related biomolecules present in liquid samples.

[0043] In an embodiment, the non-biological entities include microparticles and nanoparticles; microplastics and nanoplastics; environmental pollutants and contaminant; particulate matter such asPM10, PM2.5 and ultrafine particles; heavy metal particles; industrial microparticles including polymeric, metallic, ceramic or composite debris; fibers and synthetic fragments; and other non-biological particulate species present in environmental, industrial, food, cosmetic, pharmaceutical, or water-related samples, including air, water, wastewater, soil, and agricultural suspensions.

[0044] In an embodiment, the microfluidic device is configured for applications in environmental monitoring, including detection and analysis of pollutants and contaminants; Biomedical diagnostics, including early detection and monitoring of diseases; Material sciences, including particle sorting and characterization; Pharmaceutical development, including drug discovery and screening.

[0045] The present disclosure relates to a microfluidic device for liquid sample processing comprising an inlet 1, an outlet 2, a chamber 3, a feeding channel 4 and a collection channel 5, fluidically connected, wherein: the inlet 1, feeding channel 4, chamber 3, and collection channel 5 are arranged at successive vertical levels such that the chamber 3 is positioned below the inlet 1 and the collection channel 5 is positioned below the chamber; and wherein the chamber 3 comprises a fibrous polymeric substrate 6. The term 'comprise' is understood as indicating that the chamber is configured to receive the fibrous polymeric substrate 6.

[0046] In an embodiment, the device has a vertically stacked structure formed by at least three superposed layers, wherein: a first uppermost layer comprises the inlet 1 and the feeding channel 4, wherein feeding channel 4 extends from the inlet 1 to the chamber 3; a second layer, located below the first layer, comprises the chamber 3; a third layer, located below the second layer, comprises the collection channel 5, wherein the collection channel 5 extends from the chamber 3 to the outlet 2; and wherein the outlet 2 extends vertically through the third and second layers from the collection channel 5 into the first layer.

[0047] In an embodiment, the device comprises polydimethylsiloxane.

[0048] In an embodiment, the first, second and third layers are made of mutually different materials.

[0049] In an embodiment, the fibrous polymeric 6 substrate has a pore size ranging from 0.0003 pm to 30 pm and a porosity ranging from 2% to 59.9%, measured by image processing from scanning electron microscopy images; preferably a pore size ranging from 0.05 pm to 25 pm and a porosity ranging from 2% to 15%; more preferably a pore size ranging from 5 pm to 20 pm.

[0050] In an embodiment, the fibrous polymeric substrate is a woven or nonwoven polymeric mesh, preferably an electrospun mesh.

[0051] In an embodiment, the fibrous polymeric substrate comprises a polymer selected from polycaprolactone (PCL), polymethyl methacrylate (PMMA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyethylene oxide (PEO), polyglycolic acid (PGA), collagen, gelatin, chitosan, cellulose, cyclic olefincopolymer (COC), or mixtures thereof; preferably polycaprolactone (PCL), polymethyl methacrylate (PMMA), or mixtures thereof.

[0052] In an embodiment, the surface of the fibrous polymeric substrate is activated by plasma treatment, chemical grafting, surface oxidation, crosslinking, or incorporation of active agents.

[0053] In an embodiment, the fibrous polymeric substrate further comprises one or more capture agents selected from a list comprising antibodies, aptamers, ligand-binding proteins, peptides, nucleic-acid- based affinity probes, or combinations thereof, configured to selectively bind biological entities of interest, including circulating tumor markers, circulating tumor cells, extracellular vesicles, or disease- associated biomarkers. In a preferred embodiment, the capture agents comprise antibodies directed against one or more extracellular vesicle (EV) tetraspanins selected from CD63, CD81, CD9, individually or in combination. In a further preferred embodiment, the capture agents comprise antibodies directed against colorectal-cancer-associated circulating tumor markers, including EpCAM, EGFR, CEA, MUC1, TROP2, individually or in combination. In an optional embodiment, the capture agents further comprise antibodies for epithelial-mesenchymal transition (EMT) or sternness-associated markers, selected from CD44, Vimentin, N-cadherin, individually or a combination thereof. In another embodiment, mixed capture-agent panels combining EV tetraspanins and tumor-associated markers may be co-immobilized on the fibrous polymeric substrate to enable multiplex isolation of heterogeneous tumor-derived vesicles and circulating tumor cells.

[0054] In an embodiment, the fibrous polymeric substrate comprises a network of polymer fibers having an average diameter of 0.1 to 2 pm, measured by image analysis of scanning electron microscopy (SEM) micrographs.

[0055] In an embodiment, the device further comprises a fourth lowermost layer to seal the microfluidic device.

[0056] In an embodiment, the diameter of the inlet 1 ranges from 0.2 to 10 mm, preferably 0.5 to 5 mm, more preferably is 1 mm; and / or the depth of the inlet 1 ranges from 2 to 3 mm.

[0057] In an embodiment, the diameter of outlet 2 ranges from 0.2 to 10 mm, preferably 0.5 to 5 mm, more preferably is 1 mm; and / or the depth of the outlet 2 ranges from 5 to 8 mm, preferably is 7 mm.

[0058] In an embodiment, the diameter of the chamber 3 ranges from 1 mm to 20 mm, preferably from 2 mm to 10 mm, more preferably from 4 mm to 8 mm, even more preferably is 6.5 mm.

[0059] In an embodiment, the height of the chamber 3 ranges from 100 to 500 pm, preferably from 200 to 250 pm.

[0060] In an embodiment, the chamber 3 is bordered by a peripheral ledge with a height ranging from 100 to 500 pm, preferably from 200 to 250 pm.

[0061] In an embodiment, the length of the feeding channel 4 ranges from 1 mm to 50 mm, preferably from 3 mm to 20 mm; and / or the width of the feeding channel 4 ranges from 0.01 mm to 2 mm, preferably from 0.1 mm to 1 mm, more preferably about 200 pm; and / or the height ranges from 100 to 300 pm, preferably is 250 pm.

[0062] In an embodiment, the length of the collecting channel 5 ranges from 0.01 mm to 2 mm, preferably from 0.1 mm to 1 mm, more preferably is 0.2 mm; and / or the width of the collecting channel 5 ranges from 0.01 mm to 2 mm, preferably from 0.1 mm to 1 mm, more preferably about 200 pm; and / or a height ranging from 100 to 300 pm, preferably is 250 pm.

[0063] In an embodiment, the diameter of the fibrous polymeric substrate 6 ranges from 8 to 10 mm.

[0064] In an embodiment, the height of the microfluidic device ranges from 6 to 15 mm, preferably from 7 to 12 mm.

[0065] In an embodiment, the length of the microfluidic device ranges from 5 to 150 mm, preferably from 10 mm to 75 mm, more preferably from 35 mm to 50 mm.

[0066] In an embodiment, the liquid sample is a biological fluid, water, leachable, soil extract, soil suspension, wastewater, or airborne particulate condensate.

[0067] In an embodiment, the biological fluid is selected from blood, breastmilk, menses, saliva, or semen.

[0068] The present disclosure also relates to the use of the disclosed microfluidic device as a cell culture platform and / or drug screening platform.

[0069] An aspect of the present disclosure relates to the use of the disclosed microfluidic device as a filtration and / or isolation platform, in particular filtration and / or isolation of cells, microplastics, pollutants, or microparticles from a liquid sample.

[0070] An aspect of the present disclosure relates to the use of the disclosed microfluidic device in the in vitro diagnosis of a disease, in particular cancer, cardiovascular diseases, neurodegenerative disorders, or infectious diseases. In an embodiment, the disease is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, bladder cancer, nonHodgkin lymphoma, leukemia, cervical cancer, skin cancer (melanoma and non-melanoma), ovarian cancer, or kidney cancer.

[0071] It is also disclosed a kit comprising the disclosed microfluidic device.

[0072] The present disclosure also relates to a method to obtain the disclosed microfluidic device, wherein the method comprises the following steps:producing a first, a second and a third patterned structures using UV-photolithography to define a microfluidic channel network, comprising an inlet 1 on the first structure, a collection channel 5 on the third structure, and a chamber 3 in the second structure; casting a polydimethylsiloxane solution at a 10:1 volume ratio of pre-polymer to crosslinker onto the first, second and third patterned structures, to obtain a first, second and third layer; curing the polydimethylsiloxane for at least 30 minutes, preferably from 30 minutes to 1 hour, at 60 to 70 °C to form polydimethylsiloxane replica moldings; integrating a fibrous polymeric substrate into the chamber 3 of the microfluidic device; bonding the polydimethylsiloxane layers to assemble the microfluidic device.

[0073] An aspect of the present disclosure relates to a method for processing a liquid sample comprising a material of interest using the disclosed microfluidic device, the method comprising the following steps: introducing a liquid sample into the microfluidic device via the inlet 1; allowing the sample to flow through the microfluidic channels and interact with the fibrous polymeric substrate 6 in the chamber 3; collecting a material of interest onto the fibrous polymeric substrate 6; collecting the processed liquid sample from the outlet 2; removing the fibrous polymeric substrate 6 from the chamber 3; collecting the target molecule or cell from the fibrous polymeric substrate 6.

[0074] In an embodiment, the material of interest is selected from circulating tumor markers, circulating tumor cells, circulating extracellular vesicles, pathogenic microorganisms, disease-related biomarkers, microparticles, microplastics, pollutants, contaminants, or mixtures thereof.BRI EF DESCRI PTI ON OF THE DRAWI N GS

[0075] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention.

[0076] Figure 1: Embodiment of fiber morphology assessed by scanning electron microscopy (SEM) of electrospun fiber nets (EFNs) produced under different conditions: (A) 15% w / v polycaprolactone (PCL) dissolved in a chloroform (CHCI3) and N,N-dimethylformamide (DMF) mixture at a 7:3 volume ratio, with a flow rate of 1 mL / h and a differential potential of 12.5 kV; (B) 15% w / v PCL dissolved in CHCI3and DMF at an 8:2 volume ratio, with a flow rate of 0.7 mL / h and a differential potential of 16 kV; (C) 10% w / v PCL dissolved in CHCI3and DMF at an 8:2 volume ratio, with a flow rate of 0.7 mL / h and a differential potential of 16 kV; (D) 13% w / v PCL dissolved in CHCI3and DMF at an 6:4 volume ratio, with a flow rate of 0.9 mL / h and a differential potential of 12 kV; (E) 13% w / v PCL dissolved in CHCI3and DMF at an 7:3 volume ratio, with a flow rate of 0.9 mL / h and a differential potential of 12 kV; (F) 15% w / v PCL dissolved in CHCI3andDMF at an 6:4 volume ratio, with a flow rate of 0.9 mL / h and a differential potential of 12 kV; (G) 15% w / v polymethyl methacrylate (PMMA) (Mw 350,000) dissolved in CHCI3and DMF at an 8:2 volume ratio, with a flow rate of 1 mL / h and a differential potential of 15 kV; (H) 15% w / v polymethyl methacrylate (PMMA) (Mw 350,000) dissolved in CHCI3and DMF at an 8:2 volume ratio, with a flow rate of 1 mL / h and a differential potential of 18 kV; (I) 25% w / v polymethyl methacrylate (PMMA) (Mw 120,000) dissolved in CHCI3and DMF at an 7:3 volume ratio, with a flow rate of 0.7 mL / h and a differential potential of 17 kV;.

[0077] Figure 2: Embodiment of pore size distribution of electrospun fiber nets (EFNs) produced under conditions A-F, corresponding to the formulations described in Table 1 and the quantitative results shown in Table 2. Each condition (A-F) represents an embodiment of the fibrous polymeric substrate comprised in the disclosed microfluidic device illustrating the tunability of the EFN microarchitecture and the resulting pore size ranges (6-18 pm, 18-30 pm, 30-42 pm, 42-54 pm, and 54-90 pm).

[0078] Figure 3: Embodiment of Scanning Electron Microscopy (SEM) images of electrospun fiber nets B (EFNs-B), produced by electrospinning a 15% w / v polycaprolactone (PCL) solution in chloroform (CHCI3) and dimethylformamide (DMF) at an 8:2 volume ratio, with a flow rate of 0.7 mL / h and a differential potential of 16 kV. Following initial fabrication, EFNs-B underwent post-electrospinning treatment using polymethyl methacrylate (PMMA) dissolved in a 4:1 volume ratio solution of ethyl acetate and ethanol. Treatment variants were as follows: EFNs-B (Untreated): Electrospun sample without any additional postelectrospinning treatment; EFNs-B (PMMA 5% Treated): Electrospun sample immersed in a 5% (w / v) PMMA solution for 180 minutes; EFNs-B (PMMA 10% Treated): Electrospun sample immersed in a 10% (w / v) PMMA solution for 180 minutes.

[0079] Figure 4: Embodiment of High-Resolution Scanning Electron Microscopy (HR-SEM) image showing extracellular vesicles (EVs) bound to the surface of activated and functionalized nanofibrous substrates (EFNs). These substrates have been biofunctionalized with immobilized anti-CD63 antibodies, enabling the specific capture and isolation of EVs from the conditioned medium of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) cultured under basal conditions. The image illustrates the distribution of EVs bound to the surface of the biofunctional nanofibrous system.

[0080] Figure 5: Embodiment of Optical microscope images of electrospun fiber nets B (EFNs) composed of polymethyl methacrylate (PMMA). The images show varying PMMA molecular weights, solution concentrations, and electrospinning voltages, highlighting microparticles captured at sizes of 20 pm and 8 pm. (EFNs-G) EFNs sample with PMMA (molecular weight 350,000), 15% w / v concentration, electrospun at 15 kV; ( EFNs-H) EFNs sample with PMMA (molecular weight 350,000), 15% w / v concentration, electrospun at 18 kV; (EFNs-l) EFNs sample with PMMA (molecular weight 120,000), 25% w / v concentration, electrospun at 17 kV. Scale bar: 250 pm.

[0081] Figure 6: Embodiment of the filtering capacity of the EFN membrane (Formulation B, according to Table 1). (a) Schematic representation of the fluorescent microsphere filtration setup, (b) Capture efficiency for 8, 10, and 20 pm particles (mean ± SD, n = 3; **** p < 0.0001; ns = not significant), (c) Representative top-view fluorescence images of retained particles on the EFN membrane. Scale bar = 200 pm.

[0082] Figure 7: Schematic illustration of an embodiment of the microfluidic device design incorporating electrospun nanofibers net (EFNs): (A) Layered 2D top view diagram showing the layout of the microfluidic channels; (B) 2D lateral view diagram illustrating the vertical arrangement of the device components, including the inlet, outlet, and EFN chamber. (C) Exploded 2D top view diagram detailing each layer of the device for clarity. 1 - inlet, 2 - outlet, 3 - chamber, 4 - feeding channel, 5 - collection channel, 6 - fibrous polymeric substrate.

[0083] Figure 8: Schematic illustration of the process for producing the polydimethylsiloxane (PDMS) molds and respective layers used to produce the embodiment of the microfluidic device, including: (A) SU-8 master molds containing the inlet / outlet channels and the middle SU-8 master containing the electrospun nanofibers net (EFN) chamber; (B) EFNs cut into 8 mm diameter discs; (C) The four PDMS layers used to produce the disclosed microchip; (D) Final assembled microfluidic device embodiment. Scale bar: 10mm.

[0084] Figure 9: Photograph showing an embodiment of the microfluidic device kit components: (A) Microfluidic device incorporating electrospun nanofibers net (EFNs); (B) Tube of 1.5 mL; (C) Inlet connector; (D) Outlet connector. Scale bar: 10mm.

[0085] Figure 10: Embodiment of the operational workflow of the disclosed microfluidic device: inspection (A), setup (B), sample loading (C), washing (D), fraction collection (E) and EFN removal (F).

[0086] Figure 11: Embodiment of results of the disclosed microfluidic device compared with the commercial ScreenCell® membrane using HCT-116 cells (200,000 cells), (a) Experimental workflow for capture efficiency and downstream analysis, (b) Capture efficiency, (c) Immunofluorescence of captured EpCAM7CD45“ / DAPI+cells, (d) Metabolic activity assessed by PrestoBlue® assay, (e) Calcein-AM / PI viability of captured cells at 24, 48, and 72 h. Scale bars = 100 pm. Data represent mean ± SD (n = 3; *p < 0.05; ****p < 0.0001).

[0087] Figure 12: Embodiment of results of performance of the disclosed microfluidic device compared with the commercial ScreenCell® membrane using 240 HCT-116 cells (number selected to approximate the average CTC burden observed in metastatic patients). (A) Experimental workflow for captureefficiency assessment using inlet / outlet fractions, cytospin, immunofluorescence labeling, and downstream analyses. (B) Capture efficiency. (C) Immunofluorescence images of captured EpCAM+ / DAPr cells. (D) Metabolic activity of captured cells assessed by the PrestoBlue® assay. (E) Calcein-AM / PI viabilityof captured cells at days 1, 2, 3, and 7. Scale bars: 50 pm (C); 25 pm (E). Data represent mean ± SD (n = 3; ns = not significant; **p < 0.01; ***p < 0.001;****p < 0.0001).

[0088] Figure 13: Evaluation of CTC isolation performance using HCT-116 cells (240) spiked into healthy whole blood. Two isolation strategies were assessed: (i) density-gradient separation to obtain the mononuclear cell (MNC) fraction followed by capture on the disclosed microfluidic device, and (ii) isolation using the RosetteSep™ kit. (A) Schematic workflow illustrating spiking, both isolation approaches, and subsequent capture-efficiency assessment by cytospin and EpCAM+ / CD45“ immunolabeling. The membrane from the disclosed microfluidic device was then placed in culture for downstream analysis. (B) Representative immunofluorescence images of captured EpCAM+ / CD45“ HCT- 116 cells from cytospin analysis used to determine capture efficiency. (C) Capture efficiency. (D) Metabolic activity of cells retained on the membrane of the disclosed microfluidic device maintained in culture for 0, 7, 14, and 28 days. (E) Representative immunofluorescence images of captured EpCAM+ / CD45“ HCT- 116 cells during culture. Scale bars: 25 pm (b); 50 pm (e). Data represent mean ± SD (n = 3; ****p < 0.0001).

[0089] Figure 14: Comparative chemotherapeutic response of colorectal (HCT-116) and breast cancer (MDA-MB-231) cells in conventional 2D culture vs the disclosed microfluidic device. (A) Time-dependent viability following exposure to 5-Fluorouracil (5-FU) or Gemcitabine (GEM) quantified by PrestoBlue®. (B) Representative Calcein-AM / PI fluorescence images after 72 h drug treatment in both culture systems.

[0090] Figure 15: Embodiment of results of the capture of microplastics from environmental and biological samples using the disclosed microfluidic device. (A) Schematic workflow illustrating sample collection, homogenization, and microplastic retention using disclosed microfluidic device functionalized with different electrospun fiber network (EFN) formulations (B and C: PCL-based; H: PMMA-based); (B) Representative brightfield microscopy images showing retained microplastics on the corresponding EFN formulations.DETAI LED DESCRI PTI ON

[0091] The present disclosure relates to a microfluidic device for liquid sample processing comprising an inlet 1, an outlet 2, a chamber 3, a feeding channel 4 and a collection channel 5, fluidically connected, wherein the inlet 1, feeding channel 4, chamber 3, and collection channel 5 are arranged at successive vertical levels such that the chamber 3 is positioned below the inlet 1 and the collection channel 5 is positioned below the chamber; and wherein the chamber 3 comprises a fibrous polymeric substrate 6. The used of the microfluidic device as cell culture platform and / or drug screening platform, as a filtration and / or isolation platform, in particular filtration and / or isolation of cells, microplastics, pollutants, ormicroparticles in a liquid sample and / or also as an in vitro diagnosis platform are also disclosed. The methods to obtain the microfluidic chip and its operating workflow are also disclosed.

[0092] In an embodiment, the microfluidic device is constructed using a combination of microfluidic channels and electrospun nanofibers. The channels are designed to guide the liquid samples, both biological and non-biological, through the chip, where they interact with the biocompatible electrospun nanofiber matrix. The fibers are engineered with specific structural properties, in particular diameter, porosity, and surface functionalization, to optimize the capture of biological entities like CTMs and non- biological entities such as microparticles and environmental pollutants.

[0093] In an embodiment, the microfluidic device is used for the detection and monitoring of cancers including, but not limited to, lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, bladder cancer, non-Hodgkin lymphoma, leukemia, cervical cancer, skin cancer (melanoma and non-melanoma), ovarian cancer, kidney cancer, as well as other malignancies. Additionally, it can be applied to detect biomarkers associated with other diseases such as cardiovascular diseases, neurodegenerative disorders, and infectious diseases.

[0094] The present disclosure is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations are possible and will be apparent to those skilled in the art.

[0095] In an embodiment, the production of the disclosed microfluidic device involves several steps, including the preparation of the fibrous polymeric substrate, namely distinct electrospun fiber membranes; the fabrication of the microfluidic layers; and the integration of both components into a single functional system.

[0096] In an embodiment, the fibrous polymeric substrate (fibrous membranes) were produced by electrospinning under specific processing conditions, as detailed in Table 1. The term "fibrous membranes" is also used interchangeably with "electrospun fiber nets" in the text.Table 1 - Embodiment of processing conditions for obtaining electrospun fiber nets (EFNs), detailing the parameters used to EFNs with varying properties.Polymer Ratio Collector Voltage Flow rateA 15% PCL 7:3 (v / v) 18 12.5 1 23-27 36-47B 15% PCL 22-23 36-38. • . o.Z (V / V) zu io u. / . ..C 10% PCL 24-25 35-36D 13% PCL 6:4 (v / v)18 12 0.9 20-23 30-36E 13% PCL 7:3 (v / v)15% PCL 6:4 (v / v)15% PM MA15 350Mw8:2 (v / v) 15 . 115% PMMA18 22-23 34-37 350Mw25% PMMA7:3 (v / v) 20 17 0.7 120MwCHCL3 - Chloroform; DMF - N,N-dimethylformamide; PCL - polycaprolactone; PMMA -Poly(methylmethacrylate); T - Temperature; RH - Relative humidity.

[0097] In an embodiment, the EFN are prepared from polycaprolactone (PCL) or polymethyl methacrylate (PMMA). PCL is an aliphatic polyester formed from e-caprolactone monomer units, having a number-average molecular weight (Mn) in the range of 70,000 to 90,000 g / mol, as determined by gel permeation chromatography (GPC). PCL typically exhibits a melting temperature (Tm) between 55 °C and 65 °C and a glass transition temperature (Tg) between -60 °C and -50 °C, measured by differential scanning calorimetry (DSC). PMMA is an amorphous polymer comprising methyl methacrylate repeating units, having an Mn of 40,000 to 180,000 g / mol and an Mw of 120,000 to 350,000 g / mol (measured by GPC), with a typical Tg between 100 °C and 120 °C (measured by DSC). The dispersity (D) of each polymer is preferably between 1.5 and 2.5, measured by GPC.

[0098] In an embodiment, polycaprolactone (PCL) or polymethyl methacrylate (PMMA) were dissolved in solvent mixtures, typically consisting of chloroform (CHCI3) and N,N-dimethylformamide (DMF), and electrospun under controlled conditions to generate nanofibers with the desired properties. Key parameters such as flow rate, voltage, and collector distance can be adjusted to fine-tune the fiber morphology and optimize properties for specific applications. While PCL and PMMA were used in the examples described, other polymers such as polylactic acid (PLA), poly-L-lactic acid (PLLA), polyethylene oxide (PEO), polyglycolic acid (PGA), and natural polymers like collagen, gelatin, chitosan, cellulose, cyclic olefin copolymer (COC), or mixtures thereof, can also be used to produce the electrospun fiber nets (EFNs), allowing for customization of the EFNs' properties for specific applications.

[0099] In an embodiment, the structural properties of the EFN such as fiber diameter, porosity, and pore size can be controlled by manipulating specific process parameters: (i) solvent ratio, (ii) flow rate, (iii) electrospinning voltage, (iv) polymer type (v) tip-to-collector distance, (vi) needle internal diameter, (vii) ambient temperature, and (viii) relative humidity. Larger pore sizes resulted in higher mesh porosities. An EFNs with sufficient pore size to facilitate the isolation and capture of biological and non-biological entities within its structure is intended for insertion into the microfluidic device. Figure 1 presents an embodiment of the morphology of the EFNs produced under various electrospinning conditions, as assessed by scanning electron microscopy (SEM), showing both top-view and cross-section images of samples A-l (asdescribed in Table 1). As illustrated, variations in the electrospinning parameters generate distinct fiber morphologies and microarchitectures, including differences in fiber thickness, network density, pore size distribution, and three-dimensional organization. Samples A-F correspond to PCL-based EFNs displaying nanofibrous networks with heterogeneous porosities, whereas samples G-l correspond to PMMA-based EFNs produced from polymers of different molecular weights and concentrations, exhibiting thicker microfibers or bead-on-string structures depending on the processing conditions. The cross-section images further reveal differences in internal stacking and porosity, demonstrating the tunability of EFN microstructure for optimized filtration and capture performance.

[0100] Table 2 presents an embodiment of microarchitectural features of various EFN samples as determined by micro-computed tomography (microCT) analysis.Table 2: Embodiment of microarchitecture features of EFN samples determined by micro-computed tomography (microCT) analysis, showcasing variations in fiber diameter, pore size, and overall network structure. Statistical analysis: one-way ANOVA with the Tukey's HSD post hoc test were applied. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Polymer Ratio Mean Pore Porosity Inter-Connectivity°n ltlOn[w / v-%] [CHCI3:DMF] Size (pm) (%) (%)A 15% PCL 7:3 (v / v) 22.4 ± 6.2 46.3 ± 13.6 69.9 ± 3.6B 15% PCL 8:2 (v / v) 13.9 ± 0.8A"" 13.1 ± 0.9A"" 88.5 ± 1.1A"C 10% PCL 8:2 (v / v) 16.6 ± 0.4B" 37.1 ± 0.6B" 67.1 ± 1.2B’"D 13% PCL 6:4 (v / v) 16.1 ± 1.2A* 14.4 ± 5.6A***; C* 87.2 ± 4.1A**; C”E 13% PCL 7:3 (v / v) 13.4 ± 1.1A*"*;C" 8.2 ± 4.7A*"*'c*"*92.0 ± 4.6a*"*; C*~*F 15% PCL 6:4 (v / v) 15.2 ± 1.8A" 14.8 ± 7.5A" 86.5 ± 6.7A*; C”

[0101] An analysis of the microarchitectural parameters presented in Table 2 shows that the EFN samples exhibit distinct structural characteristics depending on the electrospinning conditions applied. Samples A, C, D, E, and F display moderate to high porosity and inter-connectivity values, compatible with networks containing broader pore size distributions. In contrast, sample B presents a significantly smaller mean pore size and substantially reduced porosity, indicating a more compact fibrous arrangement. These results demonstrate that modulation of polymer concentration, solvent ratio, and processing parameters directly influences the three-dimensional organization of the EFNs, enabling controlled adjustment of pore size and network density.

[0102] To further illustrate the architectural variations reported in Table 2, Figure 2 presents the pore size distribution profiles of EFN samples A-F, as determined by microCT analysis. The histograms quantify the proportion of pores within predefined size ranges (6-18 pm, 18-30 pm, 30-42 pm, 42-54 pm, and 54-90 pm), revealing differences in pore uniformity and heterogeneity across conditions. Samples B andC exhibit narrower distributions concentrated in smaller pore ranges, whereas samples A, D, E, and F show broader distributions with contributions from multiple pore classes. These findings complement the quantitative metrics of Table 2 and confirm that the electrospinning process allows precise tuning of pore architecture, a key factor for optimizing the capture performance of the EFNs when integrated into the microfluidic device.

[0103] In an embodiment, the fibrous polymeric membranes can be subjected to post-electrospinning treatments to further modulate their structural and surface properties. In particular, post-electrospinning treatments may include, for example, polymer infiltration or coating strategies, crosslinking, thermal annealing, solvent-vapor exposure, plasma treatment, or other chemical or physicochemical modifications intended to alter surface morphology, fiber packing density, wettability, or pore architecture of the electrospun structures.

[0104] Figure 3 depicts scanning electron microscopy (SEM) images of samples corresponding to EFNs-B (Condition B of Tabe 1), which underwent post-electrospinning treatment using 5 to 10% w / v PMMA dissolved in a volume ratio of 4:1 solution of ethyl acetate:ethanol solution. These PMMA treatments promote partial polymer deposition onto and between the fibers, resulting in surface coating, pore narrowing, and increased fiber fusion while preserving the fibrous network. As illustrated in Figure 3, these treatments produce marked changes in fiber morphology and pore architecture relative to the untreated membrane. The quantitative descriptors of each sample, including total porosity and minimum, maximum, and average pore size, as measured by imaging analysis of SEM micrographs using ImageJ, are listed in Table 3.Table 3 - Minimum, maximum and average pore size obtained by image analysis of SEM images.

[0105] This analysis shows that these structural properties can influence the capacity of the microfluidic device to effectively capture both biological and non-biological entities. In an embodiment, in all treatment conditions, including untreated (EFNs-B Untreated), 5% w / v PMMA treated (EFNs-B PMMA 5% Treated), and 10% w / v PMMA treated (EFNs-B PMMA 10% Treated), the electrospun structures maintained a minimum pore size of approximately 0.6 nm. This uniformity in minimum pore size confirms that these electrospun structures can effectively capture a range of biological and non-biological particles, making them suitable for various applications in biomedical and environmental fields.

[0106] In an embodiment, the PMMA introduced during the post-electrospinning coating step remains adhered to the fiber surfaces following solvent evaporation, forming a stable polymer layer that modifies both the surface characteristics and the pore geometry of the fibrous network. In further embodiments, the post-electrospinning coating or infiltration process may be performed using other polymers besides PMMA, including but not limited to poly(L-lactic acid) (PLLA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), cyclic olefin copolymer (COC), polyethylene oxide (PEO), chitosan, gelatin, collagen or combinations thereof. Such coatings may similarly modulate fiber surface chemistry, mechanical stability, hydrophilicity, and pore architecture, enabling tailored performance for different target applications. These surface-modifying strategies complement the structural tuning achieved during electrospinning.

[0107] In an embodiment, to further enhance interaction with target analytes, the nanofibers of the fibrous membrane can be activated.

[0108] As used herein, "activated membranes" refers to electrospun fiber nets (fibrous membranes) that have undergone a post-fabrication treatment, in particular chemical, physical, or physicochemical, to introduce or expose functional groups, reactive sites, or surface functionalities capable of enhancing their interaction with target molecules, cells, or environmental stimuli. Activation may include, for example, plasma treatment, chemical grafting, surface oxidation, crosslinking, or incorporation of active agents, resulting in improved properties such as wettability, catalytic activity, adsorption capacity, or bioactivity. An activated membrane is thus distinguished from a pristine electrospun membrane by its modified surface chemistry and enhanced functional performance.

[0109] In an embodiment for the activation of the nanofibers' surface, an ultraviolet-ozone (UV-ozone) cleaner system is used (ProCleaner™ 220, Bioforce Nanoscience). Both sides of the EFNs are exposed for 2 minutes to UV-ozone irradiation and functionalized with amine groups (-NHZ) by incubating in 1 M hexamethylene diamine (HMD) for 1 hour at 37 °C, as optimized previously

[0016] , The activated and functionalized EFNs (EFNs+) are then biofunctionalized with specific antibodies or ligands. The immobilization of these molecules on the surface of the EFNs+ is achieved using a covalent bond mediated by a coupling agent (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide / hydroxysuccinimide, EDC / NHS mixture). After incubation for 2 hours at room temperature (RT), each membrane is washed and blocked with 3% w / v bovine serum albumin (BSA) for 1 hour at RT.

[0110] For the scope and interpretation of the present disclosure it is defined that "room temperature" should be regarded as a temperature between 15-30 °C, preferably between 18-25 °C, more preferably between 20-22 °C.

[0111] In an embodiment, the maximum immobilization capacity of the system is determined using a range of antibody concentrations and the corresponding secondary antibody. An indirect quantification method is used, based on the measurement of unbound secondary antibody solution (n = 3 samples, readin triplicate) after its incubation for 1 hour at RT. Negative control samples consist of substituting the primary antibody immobilization step with 0.1 M phosphate-buffered saline (PBS).

[0112] In an embodiment of the polymeric substrate product of the present disclosure, the EDC / NHS ratio and concentrations can be optimized to enhance coupling efficiency.

[0113] In an embodiment, EDC / NHS reagents are dissolved in 0.1 M MES (2-(N- morpholino)ethanesulfonic acid) buffer with 0.9% (wt / wt) NaCI, following pH adjustment to 4.7, and mixed for 15 minutes at RT for antibody activation.

[0114] In an embodiment, a mixture of different antibodies or ligands can be immobilized on the surface of the activated and functionalized EFNs to enable the capture of a broader range of biological entities, including biomarkers associated with other diseases.

[0115] In an embodiment, EFNs have been biofunctionalized by immobilizing anti-CD63 antibodies, which enable the specific capture and isolation of EVs from the conditioned medium of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) cultured under basal conditions. Figure 4 shows a High-Resolution Scanning Electron Microscopy (HR-SEM) photograph illustrating extracellular vesicles (EVs) bound to the surface of the activated and functionalized nanofibrous substrates (EFNs). This figure demonstrates the distribution of EVs bound to the surface of the biofunctional nanofibrous system, validating the effectiveness of the antibody immobilization process in facilitating EV capture.

[0116] In an embodiment, the ability of the electrospun fiber nets (EFNs) to retain non-biological particulates was assessed using polymethyl methacrylate (PMMA) nets produced under distinct electrospinning conditions. Figure 5 presents an embodiment of optical microscope images of EFNs composed of polymethyl methacrylate (PMMA). These images illustrate the effects of varying PMMA molecular weights, polymer concentrations, and electrospinning voltages on microparticle's retention ability, according to Table 1. These variations resulted in distinct fiber packing densities and pore architectures, which directly influenced the retention of model microparticles. The particles used, fluorescent polystyrene microspheres of 20 pm and 8 pm diameter, served as surrogates for environmentally relevant microplastics, industrial particulates, and other non-biological contaminants suspended in liquid matrices. Denser and more compact fiber arrangements demonstrated increased retention of 20 pm particles, whereas more open or loosely packed structures permitted partial transit or superficial lodging of 8 pm particles. These findings demonstrate that the EFN structure can be intentionally tuned to modulate the filtration threshold, thereby enabling selective capture depending on the target application (environmental monitoring, biomedical enrichment, or pre-filtration of complex samples).

[0117] In an embodiment, to quantitatively assess the filtration performance of the EFN, controlled static gravity-driven filtration experiments were conducted using EFNs corresponding to Formulation B. Figure6 illustrates an embodiment of this characterization. As shown schematically in Figure 6a, fluorescent polystyrene microspheres with diameters of 8 pm, 10 pm, and 20 pm were suspended in aqueous solution and introduced into the upper compartment of a membrane-holding device, where the EFN was mounted horizontally to allow fluid passage by gravity. Under these conditions, the sample flowed through the EFN solely by gravitational force, enabling direct evaluation of particle retention within the fibrous network versus passage into the filtrate. These size ranges were selected to mimic the behavior of relevant biological and non-biological particulates, including red blood cells (8 pm), white blood cells (10 pm), CTCs and microplastics (20 pm), respectively.

[0118] As shown in Figure 6b, EFNs from Formulation B demonstrated high capture efficiency for 20 pm microspheres, with retention rate of 95% (mean ± SD, n = 3), while exhibiting minimal retention of 8 pm and 10 pm microspheres, which largely traversed the membrane. These findings indicate that the EFN mesh architecture allows selective retention of larger particles, such as CTCs and microplastics, while permitting the passage of smaller erythrocyte- and leukocyte-sized components. Representative fluorescence micrographs (Figure 6c) confirm this behavior, showing sparse retention of 8 pm and 10 pm microspheres at the EFN surface and pronounced accumulation of 20 pm microspheres. This selective filtration capability demonstrates the suitability of the EFNs for both biomedical and environmental applications requiring size-based discrimination.

[0119] In an embodiment, based on the structural characterization and filtration performance described above, EFNs corresponding to Formulation B were selected as the preferred membrane configuration for subsequent studies. This formulation demonstrated an optimal balance between porosity, pore size distribution, mechanical stability, and selective particle retention, making it particularly suitable for biological applications. Accordingly, Formulation B EFNs were employed in the following experiments involving capture of CTCs and integration into the disclosed microfluidic device. Their consistent performance across independent assays further supports their use as a representative embodiment of the polymeric substrate described herein.

[0120] The following embodiment describes the integration of the EFN membranes into the disclosed microfluidic device architecture. Figure 7 illustrates the schematics of an embodiment of the disclosed microfluidic device structure design by layer, incorporating EFNs in both the lateral and top view diagrams, demonstrating the integration of these nanofibers within the microfluidic channels.

[0121] In an embodiment, in a top view, the chip features an inlet 1 on the left, an outlet 2 on the right, and a central circular chamber 3 that houses the fibrous polymeric substrate 6, in particular the EFNs. In a lateral view, which reveals all the layers, the first layer includes the inlet 1 well, the second layer includes a chamber 3 to accommodate the EFNs, the third layer includes an outlet 2 well that spans along the first, second and third layer, and a fourth layer that serves as a sealing layer for the system. In a furtherembodiment, the EFNs are positioned between the second and third layers, inside the chamber 3. In an embodiment, the diameter of the inlet 1 ranges from 0.5 to 1 mm, preferably is 1mm. In an embodiment, the diameter of the outlet 2 ranges from 0.5 mm to 1 mm, preferably is 1 mm. In an embodiment, the diameter of the chamber 3 ranges from 2 mm to 10 mm, preferably from 4 mm to 8 mm.

[0122] In contrast to conventional planar microfluidic devices in which the fluid flows horizontally across laterally distributed components, the microfluidic device of the present disclosure employs a vertically stacked architecture in which the sample is conveyed sequentially through components arranged at different vertical levels. This vertical organization ensures that the entire sample volume is processed as it descends through each layer, thereby preventing sample bypass and reducing the risk of dead-volumes that typically occur in horizontal layouts. Such an arrangement is particularly advantageous for handling very small sample volumes, as it maximizes the capture and processing efficiency, improves reproducibility, and enables more accurate quantitative analysis.

[0123] The present disclosure further refers to the method for producing the microfluidic device. In an embodiment, the disclosed microfluidic device comprises at least three layers. In another embodiment, the microfluidic device is designed with four bonded layers.

[0124] In an embodiment, the method for producing different layers for the microfluidic device comprises the following steps: Produce SU-8 master molds with desired microchannel designs, lengths, and heights, including separate masters for the inlet / outlet channels and the EFNs chamber. SU-8 master mold is a precisely patterned structure created using SU-8 photoresist, acting as a template for microfabrication; Add polydimethylsiloxane (PDMS) prepared from the SYLGARD™ 184 Silicone Elastomer Kit (Dow Corning), by mixing the PDMS base (pre-polymer) with the curing agent (crosslinker) at a 10:1 ratio (w / w or v / v), and dispense the mixture onto the SU-8 master molds; Allow the PDMS to cure to obtain PDMS replica moldings with the desired microfluidic structures. In an embodiment, curing is performed at approximately 70 °C for at least 30 minutes, preferably up to 1 hour; Peel off the cured PDMS replica molds, which constitute the respective layers of the microfluidic device.

[0125] In an embodiment, the SU-8 master molds are produced using UV photolithography on silicon wafers. In a further embodiment, the polymeric substrate PDMS is added to the SU-8 master molds and allowed to cure as described.

[0126] Figure 8 illustrates an embodiment of the process of obtaining the PDMS molds and respective layers used in the microfluidic device assembly. First, the SU-8 master molds containing the inlet 1 or the outlet 2 channels and the middle SU-8 master containing the chamber 3 are obtained (Figure 8 A). EFNs are cut into 8 mm diameter discs, to be placed inside the chamber 3 (Figure 8 B). Figure 8 C depicts the four PDMS layers used to produce an embodiment of the disclosed microchip, and Figure 8 D shows the final assembled microfluidic device.

[0127] In an embodiment, the electrospun nanofibers are carefully integrated into the microfluidic channels. As an example, sample EFNs-B, as defined in Table 1, which is composed of 15% w / v PCL in a CHCI3: DM F (8:2 v / v) solvent mixture, electrospun at a flow rate of 0.7 mL / h and a voltage of 16 kV, were placed inside the chamber 3. In an embodiment, the second layer of the device comprises a recessed chamber (chamber 3) bordered by a peripheral ledge with a height of approximately 250 pm, which is dimensioned to accommodate the thickness of the EFN membrane. This ledge enables the membrane to be mechanically seated and secured between layers two and three without excessive compression, thereby preserving the native microarchitecture and pore structure of the fibers. The third layer is subsequently aligned and bonded on the bottom, effectively clamping the EFN in place while maintaining unobstructed fluid flow through the microfluidic channel network.

[0128] In an embodiment, the integration of the EFNs into the disclosed microfluidic devices achieves high-efficiency and high-purity isolation of both biological and non-biological entities.

[0129] In an embodiment, the microfluidic device of the present disclosure can be a part of a kit. Figure 9 demonstrates the content of microfluidic device kit, including the disclosed microfluidic device incorporating an electrospun nanofibers net (EFNs), one tube, such as a tube of 1.5 mL, and an inlet and outlet connectors with integrated tubing compatible with standard laboratory syringes and sample containers. Figure 10 depicts an embodiment of the operational workflow of the disclosed microfluidic device and the kit. In an embodiment, the polymeric substrate 6, namely the EFNs, can be retrieved after operating the microfluidic device and further analyzed.

[0130] In an embodiment, the microfluidic device and its corresponding kit are designed to provide a simple, user-friendly, and pump-free operational workflow. As illustrated in Figure 10, both the inlet well and the outlet well are equipped with connectors that enable rapid attachment of standard laboratory tubing. In operation, the inlet connector is coupled directly to the sample-containing tube (e.g., a 1.5 mL tube or equivalent vessel), allowing the sample to be drawn from its original container without the need for intermediate transfer steps. The outlet connector is attached to a standard syringe, which generates a gentle negative pressure to pull the sample across the microfluidic channel and through the embedded EFN membrane. This vacuum-assisted flow configuration eliminates the need for external pumps or complex instrumentation.

[0131] In an embodiment, this configuration enables complete sample processing in approximately 10 minutes per run, thereby minimizing handling time, preserving the viability of captured cells, reducing shear stress, and addressing end-user requirements for rapid and accessible sample preparation. The simplicity of the system permits operation by users with minimal technical training, including in clinical, point-of-care, field, or environmental settings.

[0132] The workflow, illustrated in Figure 10, comprises: (A) chip package inspection; (B) setup with inlet and outlet connectors; (C) sample loading into the inlet well; (D) washing of the system to remove unbound material; (E) collection of processed fractions at the outlet; and (F) retrieval of the EFN membrane for downstream analyses.

[0133] In an embodiment, the device enables quantitative assessment of capture efficiency by allowing separate collection of the inlet fraction and the outlet fraction. The inlet fraction represents the total number of biological or non-biological entities introduced into the system, whereas the outlet fraction corresponds to the non-retained entities that pass through the EFN membrane. The number of captured entities can therefore be determined directly:Captured = Inlet - Outlet and the capture efficiency can be expressed as:Capture Efficiency (%) = [(Inlet - Outlet) / Inlet] x 100

[0134] In an embodiment, this configuration ensures precise and reproducible quantification of retained targets and enables standardized analytical validation for both biological applications (e.g., CTCs, EVs) and non-biological or environmental applications (e.g., microplastics, particulate contaminants).

[0135] Surprisingly, the EFN membrane remains structurally intact after processing, thereby allowing a wide range of downstream analytical methods. For biological samples, these may include microscopy (optical, fluorescence, confocal, SEM), immunostaining, nucleic acid extraction, viability or proliferation assays, or molecular and proteomic profiling. For non-biological or environmental samples, analyses may include microparticle enumeration, spectroscopic identification, polymer-type fingerprinting, chemical composition analysis, or morphological characterization. Thus, although the operational workflow is identical for all sample types, the downstream analytical methods can be tailored to each specific application.

[0136] In an embodiment, the disclosed microfluidic device allows the capturing of biological entities within the range of 30 nm to 30 pm, specifically targeting CTCs [10 pm to 30 pm] and circulating extracellular vesicles (cEVs) [30 nm to 1 pm]. Figure 11 presents an embodiment of evaluating the performance of the disclosed microfluidic device in processing a suspension of 200 000 HCT-116 human colorectal carcinoma cells. Under identical operating conditions, the capture efficiency of the device was approximately 96%, whereas the benchmark ScreenCell® membrane (control condition) exhibited a retention of approximately 83%. These measurements were obtained by quantifying the inlet fraction and the outlet fraction through direct counting of HCT-116 cells, and calculating the number of captured cells using the capture efficiency formula previously defined. Fluorescence microscopy confirmed successful retention and preserved morphology of the captured cells. Staining for the epithelial cell adhesion molecule (EpCAM, FITC-conjugated) and nuclear counterstaining with DAPI demonstrated that the EFNmembrane supports intact cell architecture after filtration. Viability assays using Calcein-AM and Propidium Iodide, complemented with a PrestoBlue® metabolic activity assay, indicated that cells recovered from the device remained viable and metabolically active for at least 72 hours in vitro, displaying clear evidence of proliferation when maintained under culture conditions.

[0137] In contrast, cells retained on the ScreenCell® membrane did not survive under the same postcapture culture conditions. This observation shows that the mechanical and structural properties of the disclosed microfluidic device, namely its use combined with the EFN-based filtration layer, namely its compliant fibrous network, reduced shear stress, and improved surface compatibility, provide a more favorable microenvironment for maintaining viability of retained cells. The ability to preserve living cells allows downstream molecular and functional studies, including gene expression analysis, immunocytochemistry, and live-cell assays, thereby expanding the analytical potential of the device.

[0138] Figure 12 illustrates an embodiment assessing the retention and recovery performance of the device using a low-abundance population of 240 HCT-116 cells, simulating clinically relevant rare-cell conditions. Due to the low number of cells in the assay, quantification of the inlet and outlet fractions was performed using a Cytospin-based concentration step followed by EpCAM immunostaining and manual enumeration under fluorescence microscopy. This approach ensured sensitive and accurate detection of individual cancer cells in both fractions. Under these conditions, the disclosed microfluidic device demonstrated consistent capture efficiency and reliable recovery of retained cells, whereas the ScreenCell® membrane displayed lower retention and higher variability between replicates.

[0139] Fluorescence imaging of the EFN membranes following processing confirmed the presence of intact captured cells, with clear nuclear and cytoplasmic staining patterns. The structural organization of the EFNs facilitated uniform distribution of captured cells across the membrane surface, avoiding aggregation or trapping at channel edges, which is commonly observed in rigid commercial filter membranes. These results demonstrate that the disclosed device maintains high performance across a wide dynamic range of input cell concentrations, from hundreds of cells to hundreds of thousands, supporting its applicability in scenarios involving rare-event analysis, liquid biopsy, or environmental monitoring requiring high capture sensitivity.

[0140] The combined findings from Figures 11 and 12 confirm that the disclosed microfluidic device achieves superior retention of HCT-116 cells while preserving their viability and structural integrity, even under low-abundance conditions. These results highlight the advantages conferred by the use of the disclosed microfluidic device, combined with the compliant electrospun nanofiber architecture, and support its use in biological workflows requiring gentle handling and downstream analysis of captured cells. As depicted in Figures 11 and 12, retention of HCT-116 cells was superior when using the disclosed microfluidic device. Surprisingly, also cell viability was superior on cells recovered from the disclosedmicrofluidic device as compared to the commercial membrane Screencell®. Higher cell recovery from the polymeric substrate provides a significant advantage in downstream biological analysis and experimental reproducibility. By enabling a greater proportion of viable cells to be detached and collected, the system allows for more robust quantitative studies, including gene expression profiling, viability assays, and functional tests that require sufficient cell numbers to generate reliable data. Improved recovery also reduces sample loss and variability, which is particularly critical when working with scarce or sensitive cell populations. Furthermore, the ability to harvest more cells from the support broadens the range of possible follow-up experiments, facilitates longitudinal studies on the same initial culture, and enhances the overall efficiency of the workflow by maximizing the usable biological material obtained from each run.

[0141] In an embodiment, to test the feasibility of the device on a clinical setting, a sample comprising a cell number similar to the average CTC burden observed in metastatic patients was used. As depicted in Figure 12, also in this setup cell retention and viability was superior when the microfluidic device was applied, further demonstrating its suitability for rare-cell detection and processing in clinical workflows.

[0142] In an embodiment, complex matrices, namely blood, were also tested to demonstrate the applicability of the disclosed microfluidic device under clinically relevant conditions. Figure 13 illustrates an embodiment in which 240 HCT-116 human colorectal carcinoma cells were spiked into whole blood obtained from healthy donors (n > 4 independent donors). This experimental configuration simulates clinically relevant low-abundance CTC conditions. After spiking, each blood sample was divided and processed through two parallel workflows: (i) a benchmark negative-selection protocol using the commercial RosetteSep™ kit, applied to 3 mL of whole blood, in accordance with the manufacturer's instructions; and (ii) the disclosed microfluidic device, applied to the remaining whole blood, which was first processed to isolate the mononuclear cell (MNC) fraction containing the spiked tumor cells. The recovered MNC fraction (typically ~l-2 mL) was then resuspended in culture medium to restore the original whole-blood processing volume, ensuring homogeneous distribution of the spiked cells. For each microfluidic assay, 1 mL of this suspension was loaded into a single disclosed microfluidic device; experiments were performed in triplicate. An additional 1 mL aliquot of the same suspension was reserved as the inlet reference fraction, enabling accurate quantification of capture efficiency (Captured = Inlet - Outlet) as previously defined. In parallel with the disclosed microfluidic device, the benchmark RosetteSep™ negative-selection protocol was applied to 3 mL of whole blood from the same donors. Both the RosetteSep™ -enriched fraction and the fractions obtained from the disclosed microfluidic device (inlet, outlet, and EFN-retained fractions) were processed identically for downstream analysis. For all conditions, the samples were concentrated using a Cytospin-based protocol to enable sensitive detection of rare spiked cells. Following Cytospin preparation, slides were immunostained using an antibody panel compatible with CTC identification: anti-EpCAM (FITC-conjugated) for epithelial tumor cells, DAPI fornuclear counterstaining, and anti-CD45 (Texas Red-conjugated or equivalent) as the leukocyte exclusion marker. Enumeration of retained tumor cells was performed using automated image analysis (Cell Profiler or an equivalent validated computational pipeline) optimized for EpCAM+ / DAPI+ / CD45- events. This workflow ensured highly sensitive and unbiased quantification of rare spiked cancer cells in both the inlet and outlet fractions, as well as in RosetteSep™-processed samples.

[0143] As shown in Figure 13, the disclosed microfluidic device demonstrated superior retention of the spiked HCT-116 cells compared with the RosetteSep™ workflow, which exhibited lower capture efficiency and higher variability between replicates. The disclosed EFN-integrated microfluidic device consistently retained the majority of the introduced tumor cells within the EFN membrane, while maintaining minimal non-specific leukocyte retention, consistent with the size-selective properties previously demonstrated in Figure 6. To evaluate post-capture cell viability, durability, and proliferative potential, the EFN membranes containing the retained cells were transferred to culture plates and maintained under standard conditions for up to 28 days (4 weeks). Over this period, viability and metabolic activity were assessed using Calcein- AM / Propidium Iodide staining and PrestoBlue® assays at multiple time points (Day 1, 7, 14, 21, and 28). Captured cells demonstrated high initial viability, maintained epithelial marker expression, and exhibited robust proliferation, forming expanding cellular clusters during long-term culture.

[0144] Because the RosetteSep™ workflow was used exclusively for benchmarking capture efficiency, not for post-capture viability or culture, no comparison of long-term survival was performed between the two systems. Nonetheless, across all independent donors tested (n > 4), the disclosed microfluidic device consistently enabled both high recovery of rare spiked tumor cells and preservation of their long-term proliferative capacity. These findings support the suitability of the EFN-based device for downstream biological applications, including culture expansion, molecular profiling, and functional assays that cannot be performed with negative-selection systems or conventional rigid membrane filters.

[0145] In an embodiment, the disclosed microfluidic device can also function as a platform for on-chip cell culture. Owing to the compliant and porous architecture of the electrospun nanofiber membrane, captured cells remain viable, structurally preserved, and capable of adhering and proliferating directly on the EFN surface. This property enables downstream culture-based applications that are not feasible with conventional rigid filtration membranes.

[0146] In an embodiment, the disclosed microfluidic device may further function as the initial step of an integrated capture-and-culture workflow. After filtration, the EFN membrane can be retrieved from the device and transferred to standard cell-culture plates, where the retained cells continue to grow under conventional incubation conditions. Owing to the compliant, porous, and three-dimensional fibrous structure of the EFN, captured cells adhere efficiently and maintain viability, overcoming the limitations typically associated with rigid commercial membranes that do not support post-capture culture.

[0147] Figure 14 illustrates an embodiment demonstrating the capture-and-culture workflow capability using two human cancer cell lines: HCT-116 colorectal carcinoma cells and MDA-MB-231 triple-negative breast cancer cells. For each experiment, approximately 200 000 cells were suspended and processed through the disclosed microfluidic device, captured onto the EFN membrane, and subsequently transferred to standard culture wells. Parallel 2D culture controls were included. Both cell lines remained viable for at least 72 hours following capture, demonstrating that the EFN membrane provides a stable and biocompatible substrate for short-term culture independent of polymeric substrate plasticware. These findings are consistent with the viability and recovery characteristics previously demonstrated in Figures 11, 12, and 13.

[0148] Cells cultured on the retrieved EFN membranes exhibited morphology, spreading, and metabolic activity comparable to conventional 2D controls. The fibrous matrix provided a pseudo-3D microenvironment that supported adhesion and proliferation, a feature particularly advantageous for low-abundance cell populations such as CTCs. Unlike rigid filters, where cells are often unable to survive after isolation, the EFN structure preserves cell integrity and prevents apoptosis due to mechanical stress or insufficient substrate attachment.

[0149] In an embodiment to assess the use of the disclosed microfluid device, namely the retrieved EFN membrane as a platform for functional drug-response assays, captured HCT-116 and MDA-MB-231 cells were exposed to relevant chemotherapeutic agents, namely 5-Fluorouracil (5-FU) and Gemcitabine (GEM), across multiple concentrations. As shown in Figure 14a, metabolic activity measured by PrestoBlue® and viability assessed through Calcein-AM / Propidium Iodide staining demonstrated clear, dose-dependent responses for both cell lines over 24, 48, and 72 hours. These pharmacodynamic profiles were consistent with expected drug sensitivities, confirming that cells captured on the EFN membrane retain functional responsiveness.

[0150] Fluorescence microscopy (Figure 14b) further confirmed that drug-treated cells maintained characteristic nuclear and cytoplasmic staining patterns and that cytotoxicity increased proportionally with drug dose. These results demonstrate that the EFN membrane provides a reliable platform not only for maintaining cell viability after filtration but also for conducting downstream drug-screening assays on the same captured population.

[0151] Together, these findings expand the applicability of the disclosed device beyond size-based isolation. The system enables: (i) efficient capture of suspended cells, in particular cancer cells, from solution; (ii) direct transfer of the EFN membrane for on-plate culture without loss of viability; and (iii) execution of functional assays such as drug-response profiling. This multi-functionality is particularly relevant for rare CTC populations, where the preservation of even a small number of viable cells can enable downstream molecular analysis, phenotypic characterization, and personalized treatment testing.

[0152] In an embodiment, the disclosed microfluidic device can be used to test different liquid samples, namely biological fluid, water, leachable, soil extract, soil suspension, wastewater, or airborne particulate condensate. In a further embodiment, the biological fluid can be selected from blood, breastmilk, menses, saliva, semen, among others.

[0153] In an embodiment, the microfluidic device can be used to capture microplastics from environmental and biological samples. The pore size of the fibrous polymeric substrate 6 facilitates the capture of non-biological entities in the range of 20 nm to 50 pm, including microplastics in the micrometer range (for example between 1 pm and 150 pm) and airborne or waterborne pollutants such as fine particulate matter (e.g., PM2.5) and larger suspended particles. By selecting EFNs with appropriate pore architecture, the device can be tuned to preferentially retain specific size classes of microplastics and particulates while allowing smaller components of the matrix to pass through. In an embodiment, to further validate the versatility and multi-domain applicability of the disclosed microfluidic device, the system was evaluated for its ability to retain non-biological contaminants, specifically polyethylene (PE) microplastics, using electrospun fiber formulations EFNs-B, EFNs-C, and EFNs-H. Three environmentally relevant particle size ranges were assessed: 10-30 pm, 40-60 pm, and 100-140 pm, corresponding to standardized EasyMP™ batches with controlled size distributions.

[0154] In an embodiment to simulate real-world sample complexity, microplastic suspensions were mixed 50:50 (v / v) with two distinct matrices: (i) an environmental sample (freshwater), and (ii) a biological sample (human saliva). These mixtures replicated conditions typically encountered in environmental surveillance and human exposure assessments. Each 1 mL mixed suspension was processed through the disclosed microfluidic device containing EFNs-B, EFNs-C, or EFNs-H under identical operating conditions (Figure 15a). In an embodiment, the sample flow through the disclosed device was accomplished by using a standard syringe, which generates a gentle negative pressure to pull the sample across the microfluidic channel and through the embedded EFN membrane.

[0155] Following processing, the EFN membranes were retrieved and analyzed using optical bright-field microscopy (Figure 15b). Capture efficiency for each microplastic size class was assessed by direct visualization of particles retained within the fibrous network. This qualitative-quantitative approach enabled accurate determination of retention performance without the need for chemical labeling or fluorescence tagging. The results of these assays are presented in Table 4 below. EFNs-B demonstrated strong capture across all size ranges and both matrices. EFNs-C exhibited no retention of the smallest microplastics (10-30 pm) but successfully retained particles >40 pm. In contrast, EFNs-H showed efficient retention of 10-30 pm and 40-60 pm particles but did not capture the largest size range (100-140 pm).These outcomes were consistent between environmental and biological sample conditions.Table 4 - Capture performance of EFN formulations for polyethylene microplastics across size classes and matricesLegend: / minimal capture; / / moderate capture; / / / strong capture)

[0156] The micrographs presented in Figure 15b illustrate representative capture patterns, demonstrating the distribution of microplastics across the EFN surface. EFNs-B displayed dense and homogeneous particle retention, whereas EFNs-H retained microplastics in size-dependent patterns consistent with their pore architectures, as previously described in Tables 1-3.

[0157] These findings confirm that the disclosed microfluidic device, when coupled with tailored EFN formulations, provides tunable and size-selective capture of non-biological particulates across complex matrices. EFNs-B constitute an optimal broad-range filtration platform, while EFNs-C and EFNs-H enable selective retention based on particle size. This configurability extends the applicability of the device beyond biomedical workflows into environmental monitoring, exposure assessment, and contamination analysis.

[0158] In an embodiment, the high-precision microfluidic device is designed for the early detection and monitoring of various diseases, including cancers and conditions, where circulating biomarkers are relevant. The high-precision microfluidic device is applicable in clinical settings for (i) Early Disease Detection: By isolating and analyzing CTMs and other biomarkers from blood samples, facilitating early diagnosis of diseases, which is crucial for improving treatment outcomes; (ii) Personalized Medicine: The high-precision microfluidic device capability to capture CTMs, such as CTCs, culture them in vitro, and analyze specific biomarkers enables the development of personalized treatment plans based on the characteristics of a patient's condition; (iii) Environmental Monitoring: The high-precision microfluidic device can be used to detect and analyze non-biological entities such as microplastics and industrial pollutants in environmental samples, contributing to pollution assessment and public health safety.Materials and Methods

[0159] In an embodiment, unless otherwise indicated, all chemicals, polymers, buffers and solvents used in the preparation of the EFNs, biological samples, and microplastic suspensions are of analytical or cellculture grade and are obtained from commercial suppliers that are routinely used in the field, for example Sigma-Aldrich (Merck). Deionized water with a resistivity of at least 18 MO-cm is used for all aqueous solutions.

[0160] In an embodiment, PCL and PMMA used for EFN production are supplied as pellets or powder with molecular weights within the ranges, for example from Sigma-Aldrich. Solvents for electrospinning, including chloroform (CHCI3), N,N-dimethylformamide (DMF), ethyl acetate, and ethanol, are likewise obtained from commercial suppliers such as Sigma-Aldrich and are used as received or further purified if required. The PDMS used for microfluidic device fabrication is prepared from a two-part silicone elastomer kit, such as SYLGARD™ 184 Silicone Elastomer Kit (Dow Corning), mixed at a 10:1 base-to- curing-agent ratio as described. SU-8 negative photoresist and associated developers are used to fabricate the master molds for the PDMS layers.

[0161] In an embodiment, polyethylene (PE) microplastics are provided as spherical beads with defined size distributions from commercial suppliers, for example EasyMP™ batches with controlled size ranges: Batch #0297 (red, 10-30 pm), Batch #0298 (blue, 40-60 pm), and Batch #0299 (clear, 100-140 pm). Stock suspensions are supplied in aqueous / ethanolic medium containing surfactant; working suspensions are prepared by dilution in water, buffer, or the selected environmental or biological matrix immediately prior to use, followed by gentle mixing to ensure homogeneity.

[0162] In an embodiment, human cancer cell lines such as HCT-116 colorectal carcinoma cells and MDA- MB-231 triple-negative breast cancer cells are obtained from certified cell banks, for example the American Type Culture Collection (ATCC), and maintained in standard culture medium (for example, DM EM supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin) under conventional conditions (37 °C, 5% CO2, humidified incubator). Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) used for extracellular vesicle production are cultured in appropriate MSC medium under basal conditions. For all experiments, cells are used at low passages and confluency levels compatible with good cell culture practice.

[0163] In an embodiment, blood samples are obtained from healthy adult donors under appropriate institutional guidelines. Whole blood is collected into anticoagulant-containing tubes (e.g., EDTA). For experiments involving the mononuclear cell (MNC) fraction, density-gradient separation (such as Ficoll- based separation) is performed according to standard protocols to isolate MNCs, which are subsequently resuspended in culture medium as described. Saliva samples used as biological matrices for microplastic capture assays are collected from healthy volunteers, clarified by low-speed centrifugation if necessary, and mixed with microplastic suspensions at the desired ratios.

[0164] In an embodiment, electrospinning of EFNs is carried out using a single-needle electrospinning setup with a high-voltage power supply, syringe pump, and grounded collector. Polymer solutions are prepared by dissolving PCL or PMMA in the specified CHCI3:DMF ratios under stirring until complete dissolution. The solutions are loaded into syringes equipped with metallic needles, and electrospinning is performed under the conditions presented in Table 1 (flow rate, applied voltage, and tip-to-collectordistance). The resulting fiber mats are collected on suitable substrates (e.g., aluminum foil or tracing paper), dried to remove residual solvent, and stored in a desiccator until further use.

[0165] In an embodiment, structural characterization of EFNs is performed by scanning electron microscopy (SEM) and micro-computed tomography (microCT). SEM samples are sputter-coated with a thin conductive layer (e.g., Au or Au / Pd) before imaging. Pore size and fiber diameter are quantified using image-analysis software such as ImageJ / Fiji on calibrated micrographs. MicroCT data are reconstructed using the instrument's proprietary software, and porosity, inter-connectivity, and pore size distribution are extracted using dedicated image-analysis pipelines.

[0166] In an embodiment, surface activation and biofunctionalization of EFNs was carried out. Briefly, membranes are exposed to UV-ozone or equivalent activation treatment, incubated with amine- introducing reagents (e.g., hexamethylene diamine), and then subjected to carbodiimide chemistry (EDC / NHS) to covalently immobilize primary antibodies or ligands. Representative antibodies (for example, anti-CD63, anti-EpCAM, and anti-CD45) and blocking agents such as bovine serum albumin (BSA) are obtained from commercial suppliers, including Sigma-Aldrich or equivalent vendors.

[0167] In an embodiment, filtration experiments with fluorescent microspheres, cell suspensions, blood- derived samples, or microplastic-containing matrices are performed using the disclosed microfluidic device under the operational conditions described. For each assay, defined input volumes (e.g., 1 mL) are loaded through the inlet using a syringe connected to the outlet to generate negative pressure. Inlet and outlet fractions are collected separately, and the EFN membrane is retrieved at the end of the run. . Quantification is performed using one or more of the following methods, depending on the analyte: direct manual counting, immunofluorescence-based enumeration (e.g., EpCAM+ / DAPl7CD45“ for CTCs), or particle visualization under optical microscopy. For assays involving rare-cell detection or high-throughput enumeration, images are analyzed using an automated computational pipeline (e.g., CellProfiler-based workflow) optimized to identify and count target-specific markers, ensuring sensitive, reproducible, and unbiased quantification.

[0168] In an embodiment, viability and metabolic activity of captured cells are quantified using standard live / dead and metabolic assays. Representative reagents include Calcein-AM and Propidium Iodide (PI) for live / dead discrimination, and PrestoBlue® or equivalent resazurin-based assays for metabolic activity, obtained from commercial suppliers such as Thermo Fisher Scientific or Sigma-Aldrich. Fluorescence microscopy is used for qualitative and semi-quantitative assessment, whereas plate-reader measurements of fluorescence or absorbance provide quantitative readouts of metabolic activity over time. For drug-response studies, cells retained on EFNs or cultured in 2D are exposed to defined concentrations of chemotherapeutic agents, such as 5-Fluorouracil (5-FU) and Gemcitabine (GEM), and viability is monitored at predetermined time points.

[0169] 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. 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 for modifications thereof.

[0170] The above-described embodiments are combinable.References:1. Lusher, A.L., et al., Isolation and Extraction of Microplastics from Environmental Samples: An Evaluation of Practical Approaches and Recommendations for Further Harmonization. Applied Spectroscopy, 2020. 74(9): p. 1049-1065.2. Dehaut, A., L. Hermabessiere, and G. Duflos, Current frontiers and recommendations for the study of microplastics in seafood. TrAC Trends in Analytical Chemistry, 2019. 116: p. 346-359.3. Chu, X., et al., Occurrence and distribution of microplastics in water supply systems: In water and pipe scales. Science of The Total Environment, 2022. 803: p. 150004.4. Connal, S., et al., Liquid biopsies: the future of cancer early detection. Journal of Translational Medicine, 2023. 21(1).5. Lone, S.N., et al., Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Molecular Cancer, 2022. 21(1).6. Shin, H., et al., Single test-based diagnosis of multiple cancer types using Exosome-SERS-AI for early stage cancers. Nature Communications, 2023. 14(1).7. Yoshioka, Y., et al., Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nature Communications, 2014. 5.8. Shen, S.Y., et al., Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature, 2018. 563(7732): p. 579-+.9. Rasmussen, M.K., J.N. Pedersen, and R. Marie, Size and surface charge characterization of nanoparticles with a salt gradient. Nature Communications, 2020. 11(1).10. Prasad, S., A.K. Tyagi, and B.B. Aggarwal, Detection of inflammatory biomarkers in saliva and urine: Potential in diagnosis, prevention, and treatment for chronic diseases. Exp Biol Med (Maywood), 2016. 241(8): p. 783-99.11. Deveson, LW., et al., Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Nature Biotechnology, 2021. 39(9): p. 1115-+.12. Wang, J., et al., Towards microfluidic-based exosome isolation and detection for tumor therapy. Nano Today, 2021. 37.13. Contreras-Naranjo, J.C., HJ. Wu, and V.M. Ugaz, Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine. Lab on a Chip, 2017. 17(21): p. 3558-3577.14. Zhang, P., et al., Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip. Nature Biomedical Engineering, 2019. 3(6): p. 438-451.15. Pittman, T.W., et al., Saliva-based microfluidic point-of-care diagnostic. Theranostics, 2023. 13(3): p. 1091-1108.16. Casanova, M.R., et al., Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate (vol 6, 79, 2021). Npj Regenerative Medicine, 2023. 8(1).

Claims

C L A I M S1. A microfluidic device for liquid sample processing comprising an inlet (1), an outlet (2), a chamber (3), a feeding channel (4) and a collection channel (5), fl uidically connected, wherein: the inlet (1), feeding channel (4), chamber (3), and collection channel (5) are arranged at successive vertical levels such that the chamber (3) is positioned below the inlet (1) and the collection channel (5) is positioned below the chamber; and wherein the chamber (3) comprises a fibrous polymeric substrate (6).

2. The microfluidic device according to the previous claim wherein the device has a vertically stacked structure formed by at least three superposed layers, wherein: a first uppermost layer comprises the inlet (1) and the feeding channel (4), wherein feeding channel (4) extends from the inlet (1) to the chamber (3); a second layer, located below the first layer, comprises the chamber (3); a third layer, located below the second layer, comprises the collection channel (5), wherein the collection channel (5) extends from the chamber (3) to the outlet (2); and wherein the outlet (2) extends vertically through the third and second layers from the collection channel (5) into the first layer.

3. The microfluidic device according to any of the previous claim wherein the device comprises polydimethylsiloxane and / or wherein the first, second and third layers are made of mutually different materials.

4. The microfluidic device according to the previous claim wherein the fibrous polymeric substrate (6) has a pore size ranging from 0.0003 pm to 30 pm and a porosity ranging from 2% to 59.9%, measured by image processing from scanning electron microscopy images; preferably a pore size ranging from 0.05 pm to 25 pm and a porosity ranging from 2% to 15%; more preferably a pore size ranging from 5 pm to 20 pm.

5. The microfluidic device according to any of the previous claims, wherein the fibrous polymeric substrate is a woven or nonwoven polymeric mesh, preferably an electrospun mesh.

6. The microfluidic device according to any of the previous claims wherein the fibrous polymeric substrate comprises a polymer selected from polycaprolactone , polymethyl methacrylate, polylactic acid, poly-L-lactic acid, polyethylene oxide, polyglycolic acid, collagen, gelatin, chitosan, cellulose, cyclic olefin copolymer, or mixtures thereof; preferably polycaprolactone, polymethyl methacrylate, or mixtures thereof.

7. The microfluidic device according to any of the previous claims wherein the surface of the fibrous polymeric substrate is activated by plasma treatment, chemical grafting, surface oxidation, crosslinking, or incorporation of active agents.

8. The microfluidic device according to any of the previous claims wherein the fibrous polymeric substrate further comprises one or more capture agents selected from a list comprising antibodies, aptamers, ligand-binding proteins, peptides, nucleic-acid-based affinity probes, or combinations thereof.

9. The microfluidic device according to any of the previous claims wherein the fibrous polymeric substrate comprises a network of polymer fibers having an average diameter of 0.1 to 2 pm, measured by image analysis of scanning electron microscopy micrographs.

10. The microfluidic device according to the previous claim wherein the device further comprises a fourth lowermost layer to seal the microfluidic device.

11. The microfluidic device according to any of the previous claims wherein the diameter of the inlet (1) ranges from 0.2 to 10 mm, preferably 0.5 to 5 mm, more preferably is 1 mm; and / or the depth of the inlet (1) ranges from 2 to 3 mm.

12. The microfluidic device according to any of the previous claims wherein the diameter of outlet (2) ranges from 0.2 to 10 mm, preferably 0.5 to 5 mm, more preferably is 1 mm and / or the depth of the outlet (2) ranges from 5 to 8 mm.

13. The microfluidic device according to any of the previous claims wherein the diameter of the chamber (3) ranges from 1 mm to 20 mm, preferably from 2 mm to 10 mm, more preferably from 4 mm to 8 mm and / or the height of the chamber (3) ranges from 100 to 500 pm, preferably from 200 to 250 pm.

14. The microfluidic device according to any of the previous claims wherein the chamber (3) is bordered by a peripheral ledge with a height ranging from 100 to 500 pm, preferably from 200 to 250 pm.

15. The microfluidic device according to any of the previous claims wherein the length of the feeding channel (4) ranges from 1 mm to 50 mm, preferably from 3 mm to 20 mm; and / or the width of the feeding channel (4) ranges from 0.01 mm to 2 mm, preferably from 0.1 mm to 1 mm; and / or the height ranges from 100 to 300 pm.

16. The microfluidic device according to any of the previous claims wherein the length of the collecting channel (5) ranges 1 mm to 50 mm, preferably from 3 mm to 20 mm; and / or the width of the collecting channel (5) ranges from 0.01 mm to 2 mm, preferably from 0.1 mm to 1 mm; and / or a height ranging from 100 to 300 pm.

17. The microfluidic device according to any of the previous claims wherein the diameter of the fibrous polymeric substrate (6) ranges from 8 to 10 mm.

18. The microfluidic device according to any of the previous claims wherein the height of the microfluidic device ranges from 6 to 15 mm, preferably from 7 to 12 mm, and / or the length of the microfluidic device ranges from 5 to 150 mm, preferably from 10 mm to 75 mm, more preferably from 35 mm to 50 mm.

19. The microfluidic device according to any of the previous claims wherein the liquid sample is a biological fluid, water, leachable, soil extract, soil suspension, wastewater, or airborne particulate condensate, preferably wherein the biological fluid is selected from blood, breastmilk, menses, saliva, or semen.

20. Use of the microfluidic device described in any of the previous claims as a cell culture platform and / or drug screening platform, and / or a filtration platform, and / or isolation platform, in particular filtration and / or isolation of cells, microplastics, pollutants, or microparticles from a liquid sample.

21. Use of the microfluidic device described in any of the previous claims 1-19 in the in vitro diagnosis of a disease, in particular cancer, cardiovascular diseases, neurodegenerative disorders, or infectious diseases, preferably wherein the disease is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, bladder cancer, non-Hodgkin lymphoma, leukemia, cervical cancer, skin cancer (melanoma and nonmelanoma), ovarian cancer, or kidney cancer.

22. A kit comprising the microfluidic device described in any of the previous claims.

23. A method for processing a liquid sample comprising a material of interest using a device disclosed in any of the previous claims 1-19, the method comprising the following steps: introducing a liquid sample into the microfluidic device via the inlet (1); allowing the sample to flow through the microfluidic channels and interact with the fibrous polymeric substrate (6) in the chamber (3); collecting a material of interest onto the fibrous polymeric substrate (6); collecting the processed liquid sample from the outlet (2); removing the fibrous polymeric substrate (6) from the chamber (3); collecting the target molecule or cell from the fibrous polymeric substrate (6);24. The method according to the previous claim wherein the material of interest is selected from circulating tumor markers, circulating tumor cells, circulating extracellular vesicles, pathogenic microorganisms, disease-related biomarkers, microparticles, microplastics, pollutants, contaminants, or mixtures thereof.

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