Microfluidic devices

Abstract

Microfluidic devices and methods for their fabrication are described. The microfluidic devices comprise one or more enclosed microfluidic channels, wherein the internal surfaces of the microfluidic channels are modified through plasma activation and / or atmospheric pressure plasma jet treatment. The treated surfaces of the microfluidic devices can covalently attach or immobilise biomolecules or cells which is useful for applications such as an organ-on-a-chip device, lab-on-a-chip device, a separation device, a cell / biomarker-capture device, a diagnostic device, a synthesis device, or a sensor device.

Description

Microfluidic devicesField of the disclosure

[0001] The present disclosure relates to microfluidic devices containing one or more enclosed microfluidic channels. The internal surfaces of the microfluidic channels are modified through plasma activation and / or atmospheric pressure plasma jet treatment to afford surfaces useful in improving biomolecule immobilisation. The present disclosure also relates to methods of fabricating the microfluidic devices, their biofunctionalisation, and their uses.Background of the disclosure

[0002] Microfluidic devices (such as organ-on-a-chip systems) are gaining widespread use for their ability to accurately model organ-level function, separating biomolecules from bodily fluids, or as analytical tools for sensing applications. Traditionally, such microfluidic devices are constructed using a polydimethylsiloxane (PDMS) chip and a glass coverslip, though these materials lack the bioactive properties necessary to support cell growth under fluid flow.

[0003] PDMS is low cost, simple to make, flexible, non-toxic, and offers a porous matrix for gas permeation / permeability required for cell survival. Despite its advantages, its hydrophobic and inert nature is not amenable to biomolecule immobilization and hence cell attachment. Even for glass, the surface physically absorbs the biomolecules, but does not preserve them under flow and they can detach from the surface during culture or any washing steps that follow. Hence, appropriate modifications of a microfluidic channel to form robust biomimetic surfaces are sought for the successful biological application of a microfluidic device.

[0004] Current biofunctionalization approaches involve protein immobilisation using oxygen (O2) plasma treatment, and / or using coupling reagents like amino silanes. However, O2 plasma treatments are unstable, showing rapid decreases in wettability post-treatment and they do not promote the covalent immobilisation of biomolecules on the material surface. Wet-chemistry approaches such as silanization of the microfluidic channel surfaces employ reagents that are usually toxic, expensive, and involve timeconsuming techniques.

[0005] Such wet-chemistry approaches typically include incubation of silane reagents like (3-aminopropyl)triethoxysilane (APTES) and (3-aminopropyl)trimethoxysilane (APTMS) to form a covalent bond to the microfluidic device and provide amino tail groups at the channel surface. These amino groups can covalently bind to activated carboxylic groups of proteins and offer a controlled and oriented immobilization. Activation of the carboxylic group of the biomolecule is usually performed with carbodiimide chemistry. The most used carbodiimide is 1-ethyl-3-(-3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), which is often combined with N-hydroxy succinimide (NHS) to increase the conjugation efficiency. These wetchemistry approaches remain limited due to their high cost, toxicity, and the organic waste that they produce.

[0006] Therefore, the development of a microfluidic cell culture device requires precision engineering of the microfluidic channel surfaces to impart bio-functionality. This is because most mammalian cells are adherent and must attach and spread on an underlying extracellular matrix (ECM)-like surface to perform normal growth, differentiation, and metabolism.

[0007] For these reasons, the development of microfluidic devices with improved biomolecule immobilisation has become an area of increasing interest.

[0008] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the disclosure

[0009] Disclosed herein are new microfluidic devices and methods of their fabrication. The microfluidic devices comprise one or more enclosed microfluidic channels defined between an upper plate and lower plate, wherein a first portion of the internal surface of the enclosed microfluidic channel comprises a surface film, said surface film comprising carbon, nitrogen and oxygen and a second portion of internal surface being substantially free of nitrogen. Both the surface film and second portion of internal surface have a modified elemental composition relative to the upper and lower plate bulk elemental composition. Cells, biomolecules, or small organic molecules may becovalently attached to or immobilised to the surface film and / or second portion of internal surface.

[0010] In one aspect, the present disclosure provides a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein a first portion of internal surface of the one or more enclosed microfluidic channels comprises a surface film having a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said surface film comprising at least carbon, nitrogen and oxygen; and wherein a second portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said second portion of internal surface being substantially free of nitrogen.

[0011] In embodiments, the surface film has a thickness of less than about 20 nm.

[0012] In embodiments, the surface film has a thickness from about 1 to about 10 nm. Preferably from about 4 to about 9 nm.

[0013] In embodiments, the surface film has an elemental nitrogen content from about 10 to about 30 atomic % as measured by XPS.

[0014] In embodiments, the surface film has an elemental oxygen content from about 10 to about 45 atomic % as measured by XPS.

[0015] In embodiments, the surface film has an elemental carbon content from about 10 to about 40 atomic % as measured by XPS.

[0016] In embodiments, the second portion of said internal surface has an elemental nitrogen content that is less than about 20 atomic %, or less than about 15 atomic %, or less than about 10 atomic %, or less than about 5 atomic %, or less than about 1 atomic %, as measured by XPS.

[0017] In embodiments, the second portion of said internal surface has an elemental oxygen content from about 45 to about 85 atomic % as measured by XPS.

[0018] In embodiments, the second portion of said internal surface has an elemental carbon content from about 5 to about 35 atomic % as measured by XPS.

[0019] In embodiments, a majority of the internal surface of the one or more enclosed microfluidic channels comprises the surface film.

[0020] In embodiments, a minority of the internal surface of the one or more enclosed microfluidic channels is defined by the second portion of internal surface.

[0021] In embodiments, the surface film is a plasma activated coating (PAC).

[0022] In embodiments, the second portion of internal surface of the one or more enclosed microfluidic channels has been subjected to treatment by an atmospheric pressure plasma jet (APPJ).

[0023] In another aspect, the present disclosure provides a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein at least one portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said at least one portion of internal surface being substantially free of nitrogen; wherein the at least one portion of internal surface has been subjected to treatment by an atmospheric pressure plasma jet (APPJ).

[0024] In embodiments, the upper plate and the lower plate comprise different materials.

[0025] In embodiments, the upper plate and the lower plate comprise the same material.

[0026] In embodiments, the upper plate and the lower plate independently comprise one or more of metal, glass, synthetic polymer, biopolymer, quartz, ceramic, hydrogels, metal oxides, and silicon. Preferably, the upper plate and lower plate independently comprise glass and / or synthetic polymer.

[0027] In embodiments, the glass comprises one or more of silicate glass, borosilicate glass, and soda-lime glass.

[0028] In embodiments, the synthetic polymer comprises one or more of PDMS, PTFE, polycarbonate, polycaprolactone, polysulfone, polyurethane, polyethylene terephthalate, polyethylene, poly(methacrylate), polymethyl methacrylate, styrene-ethylene-butylene- styrene (SEBS), polylactic acid, polyglycolide, poly lactic-co-glycolic acid (PLGA), polystyrene, nitrocellulose, nitrocellulose blends, nitrocellulose-cellulose acetate, and cyclic olefin copolymer. Preferably, the synthetic polymer comprises PDMS.

[0029] In embodiments, the biopolymer comprises one or more of silk, alginate, collagen, gelatin, agarose, chitosan, dextran, polyhydroxyalkanoate (PHA), polylactic acid (PLA), starch and cellulose.

[0030] In embodiments, the hydrogel comprises one or more of alginate, agarose, collagen, gelatin, gelatin methacrylate (GelMA), hyaluronic acid (HA), fibrin, polyacrylamide (PAA), and polyethylene glycol (PEG).

[0031] In embodiments, the upper plate and the lower plate are bonded together.

[0032] In embodiments, the bond strength between the upper plate and the lower plate is greater than about 200 kPa.

[0033] In embodiments, the one or more enclosed microfluidic channels further comprise a membrane configured within the one or more enclosed microfluidic channels so as to define two channels separated by the membrane.

[0034] In another aspect, the present disclosure provides a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein at least one portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said at least one portion of internal surface being substantially free of nitrogen; wherein the at least one portion of internal surface has been subjected to treatment by an atmospheric pressure plasma jet (APPJ); andthe upper and / or lower plate comprise glass, synthetic polymer or a combination thereof.

[0035] In embodiments, a surface of the membrane has a modified elemental composition relative to the elemental composition of the bulk membrane.

[0036] In embodiments, the membrane comprises one or more of PDMS, polycarbonate, polyethylene, polyethylene terephthalate, polysulfone, polystyrene, parylene, polycaprolactone, polyethylene glycol diacrylate, polyurethane, polymethyl methacrylate, cyclin olefin copolymer and polyamide. In preferred embodiments, the membrane comprises one or more of PDMS, polycarbonate, polyethylene and polyethylene terephthalate.

[0037] In embodiments, the microfluidic device further comprises one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the surface film and / or second portion of internal surface.

[0038] In embodiments, the microfluidic device further comprises one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the membrane and / or membrane surface.

[0039] In embodiments, the one or more biomolecules comprise one or more proteins, polypeptides, polysaccharides, polynucleotides, nucleotides, oligonucleotides, nucleic acids, enzymes, coenzymes, antibodies, drug molecules, hydrogels, antioxidants, growth factors, vitamins, lipids, glycosaminoglycans or combination biomolecules thereof.

[0040] Non-limiting combination biomolecules include lipoproteins, glycoproteins, a nucleic acid conjugated to polyethylene glycol (PEG), a nucleic acid conjugated to a second nucleic acid, or a nucleic acid conjugated to a protein aptamer. Other combination biomolecules are contemplated.

[0041] In another aspect, the present disclosure provides a method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of:a) providing an upper plate having one or more open microfluidic channels defined therein; b) depositing a plasma enhanced chemical vapour surface film, said surface film comprising at least carbon, nitrogen and oxygen, in the one or more open microfluidic channels of the upper plate; c) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; d) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and e) bonding the upper plate to the lower plate, thereby producing the microfluidic device.

[0042] In embodiments, activating a surface of the upper plate and a surface of the lower plate in step c) comprises treatment with an atmospheric pressure plasma jet (APPJ).

[0043] In another aspect, the present disclosure provides a method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of: a) providing an upper plate having one or more open microfluidic channels defined therein; b) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; c) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and d) bonding the upper plate to the lower plate,thereby producing the microfluidic device and wherein activating a surface of the upper plate and a surface of the lower plate in step b) comprises treatment with an atmospheric pressure plasma jet (APPJ).In some embodiments, the upper plate and the lower plate comprise glass, synthetic polymer (preferably PDMS), or a combination thereof.

[0044] In another aspect, the present disclosure provides a microfluidic device according to any one of the herein disclosed embodiments, produced by a method according to any one of the herein disclosed embodiments.

[0045] In another aspect, the present disclosure provides a method of covalently bonding or immobilising organic small molecules or biomolecules to the surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to any one of the herein disclosed embodiments, comprising: a) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels; and b) covalently attaching one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels.

[0046] In another aspect, the present disclosure provides a method of covalently bonding or immobilising cells to surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to any one of the herein disclosed embodiments, comprising: a) depositing cells in a cell culture medium and contacting said cell culture medium with one or more of the enclosed microfluidic channels; b) covalently attaching biomolecules from the cell culture medium to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules; ora) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels and covalently attaching said one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; b) contacting a medium comprising cells with one or more of the enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules

[0047] In embodiments of the methods, the biomolecules comprise one or more of proteins, polypeptides, polysaccharides, polynucleotides, nucleotides, oligonucleotides, nucleic acids, enzymes, coenzymes, antibodies, drug molecules, hydrogels, antioxidants, growth factors, vitamins, lipids, glycosaminoglycans or combination biomolecules thereof.

[0048] Non-limiting combination biomolecules include lipoproteins, glycoproteins, a nucleic acid conjugated to polyethylene glycol (PEG), a nucleic acid conjugated to a second nucleic acid, or a nucleic acid conjugated to a protein aptamer. Other combination biomolecules are contemplated.

[0049] In another aspect, the present disclosure provides a microfluidic device according to any one of the herein disclosed embodiments for use in an organ-on-a-chip device, a lab-on-a-chip device, a separation device, a cell / biomarker-capture device, a diagnostic device, a synthesis device, or a sensor device.

[0050] In another aspect, the present disclosure provides the use of a microfluidic device according to any one of the herein disclosed embodiments in the manufacture of an organ-on-a-chip device, lab-on-a-chip device, a separation device, a cell / biomarker- capture device, a diagnostic device, a synthesis device, or a sensor device.

[0051] Advantages of the presently disclosed microfluidic devices, methods of fabrication, methods of covalently bonding or immobilising organic small molecules, biomolecules, or cells, and uses of the microfluidic devices include one or more of the following:• No pre-treatment (for example chemical or UV activation, which are necessary for wet chemistry approaches) or post-treatment (for example baking, which is necessary to anneal the bond between O2 plasma treated PDMS and glass) steps are required to produce the microfluidic device, which saves considerable time and cost.• Avoids the use of traditional organic coupling reagents (like amino silanes or carbodiimide chemistry) to achieve covalent immobilisation of organic small molecules, biomolecules and / or cells.• Direct covalent immobilisation of biomolecules is possible by simply contacting a protein solution with the device for a short incubation time (<1 hour).• Improved stability.

[0052] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0053] Figure 1 . A and B), XPS survey spectra of PDMS and glass respectively before and after different plasma treatments, UT denotes untreated surfaces. C) Deconvoluted C1s peaks of PAC-treated PDMS, APPJ-treated glass and O2 plasma-treated PDMS. D) C1s deconvoluted peaks of untreated surfaces. E) ESR spectra of PDMS after different treatments, x-axis represents intensity (a.u) and the y-axis represents the magnetic field (mT).

[0054] Figure 2. Shows static water contact angle and its recovery on A) PDMS and B) glass after different plasma treatments. Data displays mean ± SEM (n=3).

[0055] Figure 3. Shows the amount of immobilised fibronectin on materials with three different plasma treatments compared to A) the untreated PDMS and B) glass, both before and after SDS-washing. In Figures 3A and 3B, the bars from left to right represent untreated, O2 plasma, PAC and APPJ surfaces. Data were normalized by subtracting the fluorescence of the corresponding plasma-treated base materials. Data are mean ± SD (n = 3). * denotes P<0.05 compared to the untreated control analysed by one-way AN OVA.

[0056] Figure 4. Graph comparing cell attachment on plasma-treated PDMS and glass surfaces. TC represents tissue culture plastic. From left to right the PDMS and Glass bars represent untreated, O2 plasma, PAC and APPJ surfaces respectively. Error bars represent mean ± SD (n=9). Statistics were calculated using a two-way ANOVA and Bonferroni’s post-hoc analysis. * P < 0.05, ** P < 0.01.

[0057] Figure 5. A-B) Graphs comparing cell proliferation on plasma-treated PDMS and glass surfaces relative to tissue culture control after 3 (A) and 5 (B) days. From left to right the PDMS and Glass bars represent O2 plasma, PAC and APPJ surfaces respectively. Error bars represent mean ± SD (n=9). Statistics were calculated using a two-way ANOVA and Bonferroni’s post-hoc analysis. * P < 0.05, ** P < 0.01.

[0058] Figure 6. A-C) Graphs of cell coverage (A), CD31 levels (B), and eNOS levels (C) in microfluidic devices with various plasma treatments. Dotted or grey bars indicate the presence of fibronectin (Fn). Error bars represent mean ± SD (n=3 devices). Statistics were calculated using a two-way ANOVA and Bonferroni’s posthoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0059] Figure 7. A) General schematic of hybrid device assembly process. B) Bar graph of cell coverage on the top and bottom surfaces of O2 Plasma (left bar) and Hybrid Devices (right bar). Error bars represent mean ± SD (n=3 devices). Statistics were calculated using a one-way ANOVA and Bonferroni’s posthoc analysis. * P < 0.05, ** P < 0.01. C) Quantification of HCAEC alignment in the direction of flow across low and high wall shear stress (WSS) devices, on both surfaces. Top surface is the left bar and bottom surface is the right bar. Error bars represent mean ± SD (n=3 devices). Statistics were calculated using a two-way ANOVA and Bonferroni’s posthoc analysis. * P < 0.05, **** P < 0.0001.

[0060] Figure 8. Plot showing the mean fluorescence intensity of FITC-collagen (type I) after the SDS wash on the top PDMS surface and bottom glass surface in a hybrid chip compared to an O2 Plasma device. Error bars represent the mean ± SD (n = 3). Statistics were calculated using one-way ANOVA with Bonferroni’s posthoc analysis. * P < 0.05. Visible bar represents hybrid device.

[0061] Figure 9. Plot showing the mean fluorescence intensity of secondary goat antirabbit IgG antibody (Alexa Fluor 594) after the SDS wash on the top PDMS surface andbottom glass surface in a hybrid chip compared to an O2 Plasma device. Error bars represent the mean ± SD (n = 3). Statistics were calculated using one-way ANOVA with Bonferroni’s posthoc analysis. Most visible bar represents hybrid device.

[0062] Figure 10. A) Plot showing the mean fluorescence intensity of secondary goat anti-rabbit IgG antibody (Alexa Fluor 594) after the PBS-T wash on the top PDMS surface and bottom glass surface in a hybrid chip (right bar) compared to an O2 Plasma device (left bar). B) Plot showing the mean fluorescence intensity of the secondary goat anti-rabbit IgG antibody (Alexa Fluor 594) bound to a rabbit anti-human eNOS primary antibody after the PBS-T wash on the top PDMS surface and bottom glass surface in a hybrid chip (right bar) compared to an O2 Plasma device (left bar). Error bars represent the mean ± SD (n = 3). Statistics were calculated using one-way ANOVA with Bonferroni’s posthoc analysis.

[0063] Figure 11. Plots showing the mean fluorescent intensity of secondary goat antirabbit IgG antibody (Alexa Fluor 594) before and after the PBS-T wash on the top PDMS surface and bottom glass surface in a hybrid chip (right bars) compared to an O2 Plasma (left bars) and APTES (middle bars) devices. Error bars represent the mean ± SD (n = 3). Statistics were calculated using one-way ANOVA with Bonferroni’s posthoc analysis.

[0064] Figure 12. plot comparing mean cell attachment quantified by nuclei per field of view. The column and the error bar represent the mean ± SEM (n=3). Statistics were calculated using 2-way ANOVA and Bonferroni’s post-hoc analysis. All comparisons are related to TO. * P < 0.05, *** P < 0.001 , **** P < 0.0001. Bars from left to right represent untreated, O2 plasma, PAG and APPJ.

[0065] Figure 13. Fibronectin on PAC-PDMS promotes SMC proliferation, and collagen promotes contractility. Plots comparing A) the cell coverage, and B) SMA expression on these surfaces after 7 days of growth. The column height and error bar represent mean ± SEM (n=3). Statistics were calculated using 2-way ANOVA and Bonferroni’s post-hoc analysis. All comparisons are related to TC unless otherwise indicated. ** P < 0.01 *** P < 0.001, **** P < 0.0001. Bars from left to right represent no biomolecule, TGF-p, Collagen and Fibronectin.

[0066] Figure 14. Plots comparing A) the cell coverage and B) SMA expression on these surfaces after 7 days of growth. The column and error bar represent the mean ± SEM (n=3). Statistics were calculated using 2-way ANOVA and Bonferroni’s post-hoc analysis. All comparisons are related to TC unless otherwise indicated. ** P < 0.01, *** P < 0.001. Bars from left to right represent no biomolecule, TGF-p, Collagen and Fibronectin.

[0067] Figure 15. Fibronectin hybrid device promotes SMC proliferation to confluency. Plots comparing the cell coverage on A) Top-PDMS surface and B) Bottom-glass surface in these devices after 5 days of growth. The columns and error bars represent mean ± SEM (n=3). Statistics were calculated using 2-way ANOVA and Bonferroni’s post-hoc analysis. All comparisons are related to 02 Plasma . ** P < 0.01 *** P < 0.001. Bars from left to right represent TGF-p, Collagen and Fibronectin.

[0068] Figure 16. APPJ treatment can create microfluidic devices with channels containing micropatterned collagen and fibronectin. A) A schematic showing the micropatterning process. Confocal images showing micropatterns of B) collagen and C) fibronectin. The scale bar is 100 pm.

[0069] Figure 17. Confluent layer of SMCs and ECs on opposite sides of the PC membrane were observed in the organ-on-a-chip. A) An illustration of the bonding step to make a co-culture organ-on-a-chip. B) A micrograph of the branched region of the chip showing confluent layers of ECs and SMCs. The scale bar is 250 pm. C) SEM image of the PC membrane showing uneven pore size and distribution. The scale bar is 100 pm.Detailed description of the embodiments

[0070] It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0071] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. It will be understood that the disclosure described and defined in thisspecification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0072] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0073] For the purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0075] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “a membrane surface” means one surface of a membrane or more than one surface of a membrane.

[0076] As used herein, the term “and / or”, e.g., “X and / or Y” will be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0077] As used herein, the term “about” refers to a quantity, value, dimension, size, or amount that varies by as much as 10%, 5%, 1% or 0.1 % to a reference quantity, value, dimension, size, or amount.

[0078] Throughout the present disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0079] As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

[0080] Disclosed herein are new microfluidic devices and methods of their fabrication. The microfluidic devices comprise one or more enclosed microfluidic channels defined between an upper plate and lower plate, wherein a first portion of the internal surface of the microfluidic channel comprises a surface film, said surface film comprising carbon, nitrogen and oxygen and a second portion of internal surface being substantially free of nitrogen. Both the surface film and second portion of internal surface have a modified elemental composition relative to the upper and lower plate bulk elemental compositions. Cells, biomolecules, or small organic molecules may be covalently attached to or immobilised to the surface film and / or second portion of internal surface.Microfluidic devices

[0081] The present disclosure relates to a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein a first portion of internal surface of the one or more enclosed microfluidic channels comprises a surface film having a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said surface film comprising at least carbon, nitrogen and oxygen; and wherein a second portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said second portion of internal surface being substantially free of nitrogen.

[0082] As used herein, “substantially free of nitrogen” means an elemental nitrogen content less than about 20 atomic %, or less than about 15 atomic %, or less than about 10 atomic %, or less than about 9 atomic %, or less than about 8 atomic %, or less thanabout 7 atomic %, or less than about 6 atomic %, or less than about 5 atomic %, or less than about 4 atomic %, or less than about 3 atomic %, or less than about 2 atomic %, or less than about 1 atomic%, as measured by XPS.

[0083] The surface film may have a thickness of less than about 20 nm.

[0084] The surface film may have a thickness from about 1 to about 10 nm, or from about 2 to about 10 nm, or from about 3 to about 10 nm, or from about 4 to about 10 nm, or from about 1 to about 9 nm, or from about 2 to about 9 nm, or from about 3 to about 9 nm, or from about 4 to about 9 nm, or from about 1 to about 8 nm, or from about 2 to about 8 nm, or from about 3 to about 8 nm, or from about 4 to about 9 nm. Preferably from about 4 to about 9 nm.

[0085] Thin (less than about 20 nm) surface films may be advantageous for flexible plates / substrates (for example PDMS). Flexible plates / substrates with thicker surface films may be susceptible to cracking due to the different mechanical and thermal properties of the plate / substrate and the surface film. Thicker films may reduce the optical transparency and increase autofluorescence. Thicker films may also reduce the gas permeability of the substrate (necessary for cell growth).

[0086] The surface film may have an elemental nitrogen content from about 10 to about 30, or from about 15 to about 30, or from about 20 to about 30, or from about 25 to about 30, or from about 10 to about 25, or from about 15 to about 25, or from about 20 to about 25, or from about 10 to about 20, or from about 15 to about 20 atomic % as measured by XPS.

[0087] The surface film may have an elemental oxygen content from about 10 to about 45, or from about 15 to about 45, or from about 20 to about 45, or from about 25 to about 45, or from about 30 to about 45, or from about 35 to about 45, or from about 40 to about 45, or from about 10 to about 40, or from about 15 to about 40, or from about 20 to about 40, or from about 25 to about 40, or from about 30 to about 40, or from about 35 to about 40, or from about 10 to about 35, or from about 15 to about 35, or from about 20 to about 35, or from about 25 to about 35, or from about 30 to about 35, or from about 10 to about 30, or from about 15 to about 30, or from about 20 to about 30, or from about 25 to about 30, or from about 10 to about 25, or from about 15 toabout 25, or from about 20 to about 25, or from about 10 to about 20, or from about 15 to about 20, or from about 10 to about 15 atomic % as measured by XPS.

[0088] The surface film may have an elemental carbon content from about 10 to about 40, or from about 15 to about 40, or from about 20 to about 40, or from about 25 to about 40, or from about 30 to about 40, or from about 35 to about 40, or from about 10 to about 35, or from about 15 to about 35, or from about 20 to about 35, or from about 25 to about 35, or from about 30 to about 35, or from about 10 to about 30, or from about 15 to about 30, or from about 20 to about 30, or from about 25 to about 30, or from about 10 to about 25, or from about 15 to about 25, or from about 20 to about 25, or from about 10 to about 20, or from about 15 to about 20, or from about 10 to about 15 atomic % as measured by XPS.

[0089] The second portion of said internal surface may have an elemental nitrogen content that is less than about 20 atomic %, or less than about 15 atomic %, or less than about 10 atomic %, or less than about 5 atomic %, or less than about 1 atomic %, as measured by XPS.

[0090] The second portion of said internal surface may have an elemental oxygen content from about 45 to about 85, or from about 55 to about 85, or from about 65 to about 85, or from about 75 to about 85, or from about 45 to about 75, or from about 55 to about 75, or from about 65 to about 75, or from about 45 to about 65, or from about 55 to about 65, or from about 45 to about 55 atomic % as measured by XPS.

[0091] The second portion of said internal surface may have an elemental carbon content from about 5 to about 35, or from about 10 to about 35, or from about 15 to about 35, or from about 20 to about 35, or from about 25 to about 35, or from about 30 to about 35, or from about 5 to about 30, or from about 10 to about 30, or from about 15 to about 30, or from about 20 to about 30, or from about 25 to about 30, or from about 5 to about 25, or from about 10 to about 25, or from about 15 to about 25, or from about 20 to about 25, or from about 5 to about 20, or from about 10 to about 20, or from about 15 to about 20, or from about 5 to about 15, or from about 10 to about 15, or from about 5 to about 10 atomic % as measured by XPS.

[0092] XPS (X-ray photoelectron spectroscopy) is a surface-sensitive quantitative spectroscopic technique based on the photoelectric effect that can identify the elementsthat exist within a material (elemental composition) or that are covering its surface, as well as their chemical state, and the overall electronic structure and density of the electronic states in the material. XPS is a powerful measurement technique because it not only shows what elements are present, but may also show what other elements they are bonded to.

[0093] XPS typically has a depth penetration of about 10 nm and so may detect elements within the bulk substrate when thin surface films are present. For this reason, measurement of the elemental composition by XPS is contemplated for both the surface (consisting of the surface film and modified second portion of internal surface) of the microfluidic channels and for the surface plus bulk material from the upper and / or lower plate.

[0094] Methods to measure both the composition of the surface film, or modified portion of internal surface alone by XPS may comprise varying the angle of incidence in the XPS instrument to make the measurement depth more shallow, thereby no longer observing elements unique to the substrate in the XPS spectrum. An alternative method to correct the XPS data of thinner surfaces, films and coatings is contemplated if the plate comprises an element not present in the surface, film or coating. For example, a silicon free surface film on a glass plate may use the silicon present in the glass plate in such a method. This correction may be performed by removing the atomic% signal of the element which occurs in the plate alone and then reduce the signals of elements that occur both in the substrate and the surface, film or coating by a calculated value. This value is calculated by multiplying the ratio of those elements to the unique element (for example, silicon) by the atomic% value measured for the unique element. The remaining atomic% values of all other elements can then be rescaled so that the total comes to 100%.

[0095] A majority of the internal surface of the one or more enclosed microfluidic channels may comprise the surface film.

[0096] A minority of the internal surface of the one or more enclosed microfluidic channels may be defined by the second portion of internal surface.

[0097] The surface film may be a plasma activated coating (PAC).

[0098] The second portion of internal surface of the one or more enclosed microfluidic channels may be subjected to treatment by an atmospheric pressure plasma jet (APPJ).

[0099] The present disclosure may relate to a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein at least one portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said at least one portion of internal surface being substantially free of nitrogen; wherein the at least one portion of internal surface has been subjected to treatment by an atmospheric pressure plasma jet (APPJ).

[0100] The present disclosure may relate to a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein at least one portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said at least one portion of internal surface being substantially free of nitrogen; wherein the at least one portion of internal surface has been subjected to treatment by an atmospheric pressure plasma jet (APPJ); and the upper and / or lower plate comprise glass, synthetic polymer or a combination thereof.Upper plate and lower plate

[0101] The upper plate and the lower plate may comprise different materials.

[0102] The upper plate and the lower plate may comprise the same material.

[0103] The upper plate and the lower plate may independently comprise one or more of metal, glass, synthetic polymer, biopolymer, quartz, ceramic, hydrogels, metal oxides, and silicon. Other suitable materials known in the art are contemplated.

[0104] The glass may comprise one or more of silicate glass, borosilicate glass, and soda-lime glass. Other glass types known in the art are contemplated.

[0105] The synthetic polymer may comprise one or more of PDMS, PTFE, polycarbonate, polycaprolactone, polysulfone, polyurethane, polyethylene terephthalate, polyethylene, poly(methacrylate), polymethyl methacrylate, styrene-ethylene-butylene- styrene (SEBS), polylactic acid, polyglycolide, poly lactic-co-glycolic acid (PLGA) polystyrene, nitrocellulose, nitrocellulose blends, nitrocellulose-cellulose acetate and cyclic olefin copolymer. Other synthetic polymers known in the art are contemplated.

[0106] As used herein, the term “synthetic polymer” refers to any man-made polymer that is not a biopolymer.

[0107] As used herein, the term “biopolymer” refers to any polymer that is a natural polymer produced by the cells of living organisms, or a polymer produced from a monomer that is produced by the cells of living organisms.

[0108] The biopolymer may comprise one or more of silk, alginate, collagen, gelatin, agarose, chitosan, dextran, polyhydroxyalkanoates (PHA), polylactic acid (PLA), starch and cellulose. Other biopolymers known in the art are contemplated.

[0109] The hydrogel may comprise one or more of alginate, agarose, collagen, gelatin, gelatin methacrylate (GelMA), hyaluronic acid (HA), fibrin, polyacrylamide (PAA) and polyethylene glycol (PEG). Other hydrogels known in the art are contemplated.

[0110] As used herein, the term “hydrogel” refers to a biphasic material that is a mixture of porous, permeable solids and at least 10% by weight or volume of interstitial fluid composed completely or mainly by water. Both physical hydrogels (having non- covalent bonds) and chemical hydrogels (having covalent cross-linking bonds) are contemplated.

[0111] The upper plate and the lower plate may be bonded together.

[0112] The bond strength between the upper plate and the lower plate may be greater than about 200 kPa.

[0113] The upper and lower plate may comprise glass and / or synthetic polymer (preferably PDMS), or a combination thereof.Membranes

[0114] The one or more enclosed microfluidic channels may further comprise a membrane configured within the one or more enclosed microfluidic channels so as to define two channels separated by the membrane.

[0115] A surface of the membrane may have a modified elemental composition relative to the elemental composition of the bulk membrane.

[0116] The membrane may comprise one or more of PDMS, polycarbonate, polyethylene, polyethylene terephthalate, polysulfone, polystyrene, parylene, polycaprolactone, polyethylene glycol diacrylate, polyurethane, polymethyl methacrylate, cyclin olefin copolymer and polyamide. In preferred embodiments, the membrane comprises one or more of PDMS, polycarbonate, polyethylene and polyethylene terephthalate. Other membrane materials known in the art are contemplated.Microfluidic devices containing biomolecules / cells

[0117] The microfluidic devices as described in the present disclosure may further comprise one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the surface film and / or second portion of internal surface.

[0118] Without wishing to be bound by theory, it is envisaged that radicals within the surface film and / or second portion of said internal surface facilitate the covalent bonding or immobilisation of biomolecules or organic small molecules to provide biomolecules or organic small molecules covalently bonded or immobilised on the surface film and / or second portion of said internal surface in the microfluidic channels.

[0119] The microfluidic devices comprising a membrane may further comprise one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the membrane and / or membrane surface.

[0120] As used herein, the term “biomolecule” refers loosely to molecules present in organisms that are essential to one or more typical biological processes. Biomolecules may include large macromolecules (for example proteins, carbohydrates, lipids and nucleic acids), as well as small molecules (for example vitamins and hormones). The biomolecule may be a naturally occurring biomolecule, or a synthetic biomolecule.

[0121] The one or more biomolecules may comprise one or more proteins, polypeptides, polysaccharides, polynucleotides, nucleotides, oligonucleotides, nucleic acids, enzymes, coenzymes, antibodies, drug molecules, hydrogels, antioxidants, growth factors, vitamins, lipids, glycosaminoglycans or combination biomolecules thereof. Other biomolecules known in the art are contemplated.

[0122] As used herein, the term “combination biomolecules” refers a biomolecule that is conjugated to other molecules. The other molecule may be a second biomolecule or any other suitable molecule. For example, non-limiting combination biomolecules may include lipoproteins, glycoproteins, a nucleic acid conjugated to polyethylene glycol (PEG), a nucleic acid conjugated to a second nucleic acid, or a nucleic acid conjugated to a protein aptamer. The person skilled in the art would be aware of methods known in the art to prepare combination biomolecules.Method of fabricating microfluidic devices

[0123] The present disclosure further relates to a method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of: a) providing an upper plate having one or more open microfluidic channels defined therein; b) depositing a plasma enhanced chemical vapour surface film, said surface film comprising at least carbon, nitrogen and oxygen, in the one or more open microfluidic channels of the upper plate; c) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; d) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and e) bonding the upper plate to the lower plate, thereby producing the microfluidic device.

[0124] Processes for generating suitable PAC films are described in PCT / AU2022 / 051135 and PCT / AU2023 / 050317, the entirety of which are incorporated herein by reference.

[0125] Activating a surface of the upper plate and a surface of the lower plate in step c) may comprise treatment with an atmospheric-pressure plasma jet (APPJ).

[0126] Atmospheric-pressure plasma is a plasma in which the pressure approximately matches that of the surrounding atmosphere. Various forms of excitation can be used to produce said plasma such as alternating current excitation, direct current excitation, radio wave excitation and microwave excitation.

[0127] The present disclosure further provides a method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of: a) providing an upper plate having one or more open microfluidic channels defined therein; b) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; c) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and d) bonding the upper plate to the lower plate, thereby producing the microfluidic device and wherein activating a surface of the upper plate and a surface of the lower plate in step b) comprises treatment with an atmospheric pressure plasma jet (APPJ).Method of attaching biomolecules and / or cells to a microfluidic channel

[0128] The present disclosure further provides a method of covalently bonding or immobilising organic small molecules or biomolecules to the surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to any one of the herein disclosed embodiments, comprising:a) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels; and b) covalently attaching one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels.

[0129] As used herein, the term “contacting” includes flowing a liquid into or through the one or more enclosed microfluidic channels, or having the liquid remain static within the one or more enclosed microfluidic channels for a period of time.

[0130] In another aspect, the present disclosure provides a method of covalently bonding or immobilising cells to surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to any one of the herein disclosed embodiments, comprising: a) depositing cells in a cell culture medium and contacting said cell culture medium with one or more of the enclosed microfluidic channels; b) covalently attaching biomolecules from the cell culture medium to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules; or a) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels and covalently attaching said one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; b) contacting a medium comprising cells with one or more of the enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules

[0131] In embodiments of the method, the biomolecules comprise one or more of comprise one or more proteins, polypeptides, polysaccharides, polynucleotides, nucleotides, oligonucleotides, nucleic acids, enzymes, coenzymes, antibodies, drugmolecules, hydrogels, antioxidants, growth factors, vitamins, lipids, glycosaminoglycans, or combination biomolecules thereof.

[0132] In another aspect, the present disclosure provides a microfluidic device according to any one of the herein disclosed embodiments for use in an organ-on-a-chip device, a lab-on-a-chip device, a separation device, a cell / biomarker-capture device, a diagnostic device, a synthesis device, or a sensor device.

[0133] In another aspect, the present disclosure provides the use of a microfluidic device according to any one of the herein disclosed embodiments in the manufacture of an organ-on-a-chip device, lab-on-a-chip device, a separation device, a cell / biomarker- capture device, a diagnostic device, a synthesis device, or a sensor device.

[0134] The microfluidic devices may be used to immobilise extracellular matrix proteins for culturing cells, immobilise enzymes for sensing metabolites, immobilising antibodies to run immunoassays, or as a platform to isolate cells and biomolecules from bodily fluids. Other potential uses of the microfluidic devices are contemplated.ExamplesExample 1 : Materials and methodsMaterial preparations

[0135] PDMS and glass samples (12 mm x 8 mm) were independently selected for use as an upper plate and / or a lower plate, and used for surface characterisation and static cell culture analysis. For testing the covalent immobilisation of proteins, they were further cut down to 0.3 mm x 0.4 mm to fit a 96-well plate. PDMS was made by mixing the monomer and cross linker in a 10:1 ratio and degassed to remove bubbles. The degassed PDMS was then baked at 60 °C for 4 hours. Glass plates were first sonicated in acetone, isopropanol, and MilliQ water (3 x 15 seconds per solution) before being cut to size for experiments.Plasma treatments

[0136] O2 plasma treatment was performed as previously described (Ruhoff, A. M. et al. Biomaterial Wettability Affects Fibrin Clot Structure and Fibrinolysis. Advanced Healthcare Materials, 2100988 (2021)). Briefly, a plasma chamber (Harrick Plasma, PDC-002-HP) was evacuated to 50 mTorr, after which oxygen gas was introduced andthe pressure was maintained at 1.72 Torr. Samples were then plasma-treated for 1 minute at 45 W and used immediately.

[0137] Plasma activated coating (PAC) treatment was performed in a separate plasma system under a vacuum. The samples were placed on a stage inside a chamber. The stage was connected to a negative pulse generator RLIP6 (GBS Elektronik GmbH, Germany). The vacuum was applied to achieve a pressure of less than 5x10-5Torr. Before deposition of a PAC, the samples were activated using argon ions for 10 minutes at radio frequency (RF) power of 75 W and pressure maintained at 80 mTorr. The chamber was evacuated again to a pressure less than 5x10-5Torr. PAC treatment was performed by injecting a reactive gas mixture of acetylene (1 standard cubic centimeters per minute (SCCM)), nitrogen (3 SCCM), and argon (10 SCCM) into the chamber at 50 W RF power for between 6 and 10 minutes. The longer the duration of PAC treatment, the thicker the film produced. The pressure inside the chamber was maintained at 110 mTorr and the gas flow rate was controlled by setting flow rates on a mass flow controller (Allicat Scientific). In all cases, the bias applied was 500 V with a frequency of 3 kHz and a pulse length of 20 ps. After treatment, samples were immediately used for all experiments.

[0138] The atmospheric pressure plasma jet (APPJ) was generated in a glass tube with outer and inner diameters of 4.6 mm and 2.0 mm respectively. A double electrode design with two copper strips used as the powered and grounded electrodes was used. The strips were 15 mm wide and 0.1 mm thick and wrapped around the glass tube with 20 mm separation. The powered electrode was placed with its end 5 mm away from the outlet end of the glass tube. Helium, the working gas, was fed through the glass tube at a rate of 2 L / min. An in-house power supply delivered high-voltage AC sinusoidal signals. The resonant frequency was 32 kHz. The oscilloscope used to display the waveform was obtained from Tektronix (TDS-2024B). The current was measured from the ground electrode using a 100 ohm resistor (Allen Bradley 2 W, 5 % CC). APPJ was mounted on an in-house modified 3D printer (FISun i3 Prusa). The printer was operated with the Repetier software, and codes were written in-house using MATLAB. The scanning speed of the APPJ treatment was 2.50 m / min conducted in parallel lines spaced 5 mm apart in a raster fashion. The samples were used immediately after treatment.X-ray Photoelectron Spectroscopy (XPS)

[0139] X-ray photoelectron spectroscopy (XPS, Thermo ScientificTM K-Alpha spectrophotometer, ThermoFisher Scientific, Waltham, MA, USA) equipped with a monochromatic Al Ka X-ray source was used for surface characterisation. Survey spectra were acquired within the binding energy range from 0 to 1400 eV with a resolution of 1 eV and spot size of 400 pm and a total of 15 scans for each spectrum. Data were processed using Avantage software (version 5). The spectra were charge corrected by shifting the C-C / H component of C1s to 284.8 eV. Three measurements were taken per condition and representative plots are presented. The error represented is the standard error of the mean.Variable Angle Spectroscopic Ellipsometry (VASE)

[0140] The thickness of the PAC layers deposited on the upper and / or lower plate was measured using variable angle spectroscopic ellipsometry (VASE). PAC was deposited on silicon wafers and the data was collected at three angles of incidence (65°, 70°, 75°) using a J.A Woollam M2000 V spectroscopic ellipsometer. A model consisting of silicon substrate, silicon oxide layer (2 nm) and a Cauchy layer to represent the PAC was used to fit the measurements. The error represented is the standard error of the mean.Electron Spin Resonance Spectroscopy

[0141] The presence of radicals on the surface of the upper or lower plates were characterized using electron spin resonance spectroscopy (ESR, SpinScan X, Belarus). The operating frequency was 9.35 GHz at a central magnetic field of 336 mT and microwave power of 2 mW at room temperature. Instead of glass, quartz was used, as the added components in glass interfere with the microwave frequency of the instrument. Both quartz and PDMS samples were cut into 40 mm x 5 mm and subjected to different plasma treatments. Within 1 hour after the treatments, the samples were fixed to an ESR tube of diameter 2 mm and placed inside the instrument cavity. 5 scans were measured for each sample and the data was plotted using GraphPad Prism (version 10.0.1).Static Water Contact Angle

[0142] The static water contact angle was measured using 3 pL droplets from an attention Theta tensiometer (Biolin Scientific, Vastra Frdlunda, Sweden). 3 measurements for each condition were obtained.Atomic Force Microscopy

[0143] Surface topography of the plates before and after different plasma treatments were characterised using atomic force microscopy (AFM, Bruker Innova, Germany). A monolithic silicon AFM probe (Tap300 Al-G, Budget Sensors, Bulgaria) under tapping mode, was used to scan a 20 pm x 20 pm area on the samples at a 0.5-1 Hz scan rate. The force constant of the cantilever of the probe was 40 N / m, and the resonance frequency around 300 kHz. The tip radius was 10 nm. The images obtained were postprocessed using Gwyddion software (version 2.63) (Necas, D. & Klapetek, P.Gwyddion: an open-source software for SPM data analysis. 10, 181-188 (2012)). The error represented is the standard error of the mean (SEM).Biomolecule immobilization

[0144] Fibronectin was fluorescently labelled with Alexa Fluor 647 fluorophore using the Alexa Fluor™ 647 protein labelling kit (ThermoFisher Scientific, A20173) according to the manufacturer’s instructions. Briefly, sodium bicarbonate (50 pL, 1 M) was added to fibronectin (Sigma Aldrich, 11080938001) (500 pL, 2 mg / mL) and transferred to one vial of reactive dye. Contents were stir-mixed for 1 hour at room temperature. Labelled fibronectin was then purified using a column packed with purification resin.

[0145] A black 96-well plate was blocked with 5% BSA and washed 3 times with PBS prior to loading the samples. Fluorescent fibronectin (60 pg / mL) was incubated on samples at 37 °C for 1 hour. To remove non-covalently attached proteins, samples were incubated with sodium dodecyl sulphate (SDS, 5% w / v in deionized water, 300 pL) at 60 °C for 1 hour and washed 4-5 times with PBS. The relative amount of immobilized fibronectin was determined by measuring fluorescence (excitation 650 nm / emission 668 nm) using a plate reader (CLARIOstar plus, BMG LABTECH) before and after the SDS wash.Microfluidic Device Preparation and Setup

[0146] Moulds for the microfluidic channels (1000 pm width, 200 pm thickness) were fabricated using standard dry-film photolithography techniques at the Research Prototype Facility at The University of Sydney. For PDMS, after silanization with chlorotrimethylsilane (Sigma Aldrich, USA), the PDMS was cast onto the channels and cured at 60 °C for a minimum of 4 hours. Inlet and outlet holes were created using a 1 mm biopsy punch, with the upper and lower plates of the devices subsequently bonded by manual conformal contact at room temperature using the plasma treatments described previously. O2 plasma devices were baked for 15 minutes at 60 °C before use to anneal the bond. Other devices were used immediately after bonding.

[0147] For the hybrid device, custom-designed 3D-printed masks were placed over the upper plate to expose only the open microfluidic channel regions. PAC was then applied after which the mask was removed. The upper and lower plates were then both subjected to APPJ treatment and bonded together. The device was used immediately. Devices were coated with fibronectin (60 pg / mL in PBS) for 1 hour under standard incubator conditions, before washing with MesoEndo cell growth medium (Cell Applications, 212-500).Bond Strength Analysis

[0148] The bond strength of the microfluidic devices was analyzed using 3 different methods - leak, burst, and bonding tests. The leak test was performed by connecting the devices to a syringe pump and then flowing milli-Q water at a rate of 20 pL / hr for a day. A burst test was performed by pipetting milli-Q water into the inlets at a rate of 1 mL / s. A bonding strength test was performed using a universal mechanical tester (Shimadzu EZ-LX). The microfluidic device was placed in a sample holder and a T- shaped structure was glued to the top of the device. With the sample holder fixed, the T- shape was pulled upward at a constant rate of 5 mm / min and at a constant force of 5 kN. The force per unit area at the time of failure is reported as the strength. The error mentioned is the standard error of the mean (SEM).Cell Culture

[0149] Human Coronary Artery Endothelial Cells (HCAECs) (Cell Applications, 300- 05a) were cultured in MesoEndo Cell Growth Medium until 70-80% confluent. HCAECs were used between passages 3-6 for experiments. For attachment and proliferationassays, HCAECs were seeded onto samples at 10,000 cells / cm2. The attachment was measured 1 hour after seeding, with proliferation assessed after 3 and 5 days.

[0150] For microfluidic devices, HCAECs were seeded at 4-5 x 106cells / mL and incubated for 3-4 hours to enable cell attachment. Devices were connected to a syringe pump with media continuously perfused through the device for 3 days at 20 pL / hr (Wall Shear Stress - 0.007 dynes / cm2). For experiments conducted under coronary shear conditions, devices were connected to a peristaltic pump and with the flow rate progressively increased to reach 500 pL / min (Wall Shear Stress - 12.7 dynes / cm2) within two days. Devices were cultured under these shear conditions for another 3 days.Immunofluorescence

[0151] Material samples and devices were fixed for 20 minutes using 4% (v / v) paraformaldehyde in PBS and washed with PBS (3 x 5 mins). For attachment and proliferation experiments, samples were incubated with Hoechst 33342 (ThermoFisher Scientific, R37605) (35 pL / mL in PBS) for 30 minutes and washed with PBS (3 x 5 mins).

[0152] For microfluidic devices, HCAECs were permeabilized using 0.1% Triton X-100 (v / v in PBS) for 5 minutes and washed with PBS (3 x 5 mins). To prevent non-specific binding, cells were blocked with 5% (w / v) bovine serum albumin (BSA) in PBS-T (Phosphate Buffered Saline with 0.05% Tween 20) for 1 hour before incubation of the unconjugated rabbit anti-human eNOS antibody (ThermoFisher Scientific, PA1-037) overnight at 4°C in blocking buffer. Cells were subsequently washed with PBS-T (3 x 5 mins) and incubated with the secondary goat anti-rabbit IgG antibody (Alexa Fluor 594) (1:1000) for 2 hours. After washing (3 x 5 mins - PBS), cells were incubated with mouse anti-human CD31 (Alexa Fluor 488, Australian Biosearch, 303110) in a blocking buffer (1:100) overnight at 4°C. In relevant cases, samples were stained with Phalloidin (ThermoFisher Scientific, 41-6559-05) as per the manufacturer's instructions. Cells were then washed (3 x 5 mins) and stained for Hoechst 33342 as described above.Microscopy and Image Analysis

[0153] For attachment and proliferation quantification, images were captured using a Zeiss Axio Vert.AI (5x) with five images acquired per sample. Microfluidic devices wereimaged using a 10x / 0.45 NA objective lens on a Zeiss 880 confocal microscope (Zeiss, Germany).

[0154] All images were analyzed using ImageJ / Fiji (Schindelin, J. et al. Fiji: an open- source platform for biological-image analysis. Nature Methods 9, 676-682 (2012)). For attachment and proliferation, a script was developed to threshold nuclei and determine the mean nuclei count across each image; this script was applied to all acquired images using batch processing. For microfluidic devices, eNOS was quantified using mean fluorescence intensity per cell and CD31 using the integrated density per cell. Cell alignment was determined by measuring the angle between the flow direction and the major axis of each cell, converted to a percentage scale from 0% (normal orientation) to 100% (perfect parallel alignment).Statistical Analysis

[0155] Results were reported as mean ± standard deviation or mean ± SEM for a minimum of three independent experiments. Where appropriate, statistics were calculated using a one-way or two-way ANOVA with Bonferroni’s posthoc correction on GraphPad Prism (v9). Statistical significance was established at P < 0.05.Immobilisation of other ECM proteins and antibodies

[0156] Covalent attachment of antibodies and proteins was performed in the microfluidic chip. Hybrid and O2 Plasma devices were compared. FITC-conjugated bovine collagen type -I (Sigma Aldrich, C4361) at 60 pg / mL in PBS (phosphate buffered saline) was pipetted into the microfluidic channels and incubated at 37 °C for 1 hour. An SDS (5% w / v in deionized water) wash was performed for 1 hour at 60 °C, followed by 3xPBS washes, after which images of the top (PDMS) and bottom (glass) surfaces were captured using a confocal microscope (Zeiss 880). The fluorescent images were quantified for mean fluorescence intensity using Imaged. Similarly, a secondary goat anti-rabbit IgG antibody (Alexa Fluor 594,1 :1000 in PBS, 1 hour at room temperature) was immobilised in the chips, and the covalent attachment was studied.

[0157] To study the shear resistance of immobilized biomolecules in the chip, a secondary goat anti-rabbit IgG antibody (Alexa Fluor 594, 1 :1000 in PBS, 1 hour at room temperature) was immobilised and washed with PBS-T (0.05% Tween-20 in PBS) (3 x 5 minutes) at the rate of 20 pL / s. The chips were then imaged and analysed asdescribed above. Additionally, separate experiments were conducted using a rabbit anti-human eNOS primary antibody (1 :100 in blocking buffer, 1 hour at 37o C), with the antibody washed off with PBS-T (3 x 5 minutes) at 20 pL / s. The channels were blocked with 5% BSA for 1 hour at room temperature. After this, a secondary goat anti-rabbit IgG antibody (Alexa Fluor 594,1 :1000 in PBS, 1 hour at room temperature) was flushed through the channels. Washing was performed with PBS-T (3 x 5 minutes), and then confocal imaging was conducted as described previously.

[0158] The stability of immobilized antibodies in a hybrid chip was compared against that of an O2 Plasma device treated with APTES. After bonding, O2 Plasma devices were treated with APTES (2% in acetone, Sigma Aldrich 440140) and incubated for 2 hours. The channels were then washed with distilled water. Secondary goat anti-rabbit IgG antibodies (Alexa Fluor 594, 1 :1000 in PBS,1 hour at room temperature) were immobilized as above, washed off with PBS-T (3 x 5 minutes), and imaged.Models for smooth muscle cell proliferation

[0159] Human coronary artery smooth muscle cells (HCASMCs, Cell Applications, 300-05a), hereafter referred to as SMCs, were cultured in a smooth muscle cell growth medium (SmGM-2 Bullet kit, CC-3182). Cells between passages 3-6 reaching 70-80% confluency were used for experiments. Static cell attachment and proliferation were performed similarly to HCAECs, with the proliferation also measured at day 7. To study proliferation, samples were treated and coated with the following biomolecules (same protocol as before) and compared: fibronectin (60 pg / mL in PBS), collagen type IV (60 pg / mL in Hanks balanced salt solution, HBSS), transforming growth factor-p (TGF-p, 10 ng / mL in PBS).

[0160] For microfluidic culture, cell growth was compared in a hybrid device against an O2 Plasma-treated device for five days at 20 pL / hr (WSS — 0.007 dyn / cm2). The devices were coated with fibronectin, collagen, or TGF-p for one hour under standard incubator conditions. To remove excess coating, channels were washed with SMC medium by pipetting medium through the channel before culturing SMCs at a concentration of 5-6 x 106cells / mL.Co-culture Organ-on-a-chip

[0161] PC membranes (pore size 8 pm, WHA155846, Whatman Nucleopore Track- etched) were slow APPJ (S-APPJ, scan speed of 2.50 m / min) treated on both sides. They were then sandwiched between Fast APPJ (F-APPJ, scan speed of 3.60 m / min)- treated PDMS devices to form an irreversible bond. The organ-on-a-chip was then sterilized with UV for 30 minutes, and then coated with fibronectin as described above.

[0162] For the cell culture in the organ-on-a-chip, SMCs were seeded onto the top channel and cultured for two days at 20 L / hr (WSS — 0.007 dyn / cm2). ECs were seeded into the bottom channel, and the device was inverted for two to three hours to enable cell attachment to the membrane. The device was then inverted back and cultured for three days at 20 pL / hr (WSS — 0.007 dyn / cm2).Micropatterning of proteins

[0163] Silicon microfluidic wafers were prepared using standard photolithography at the Research and Prototype Foundry at the University of Sydney, and PDMS was cast as described before (original ROI). PDMS stamps of 20 pm rails and 40 pm gaps were cut off from the wafer. FITC-conjugated Type I collagen (10 pL, 1 mg / mL, Sigma Aldrich, C4361) or FITC-conjugated fibronectin (10 pL, 1 mg / mL, Sigma Aldrich, F2733) were pipetted on the stamps. The stamps were incubated in a humid chamber for 20 minutes, followed by another round of incubation for 20 minutes at ambient conditions. After this, the stamps were dried using a nitrogen gun and placed onto glass coverslips treated with S-APPJ. A 30 g weight was placed on the stamps, and the pattern transfer was allowed for 10 minutes. After this, the stamps were lifted off. An F-APPJ-treated PDMS channel was bonded to the coverslip. The channel was placed on top of the patterns so that the flow direction was perpendicular to the patterns.Example 2: Surface Characterisations

[0164] Three different plasma treatments were investigated: namely O2 plasma, PAC, and APPJ. O2 plasma uses oxygen as the working gas whereas PAC uses a discharge in a combination of acetylene and nitrogen at a negative bias, and APPJ uses a helium gas discharge that scans across the surface at a defined speed. A strong oxygen peak was evident after all the treatment processes. This appears because radicals and reactive groups readily undergo oxidation reactions with atmospheric oxygen whenexposed to air after the treatment due to their high energy. These oxidation processes are known as auto-oxidation as they occur spontaneously upon exposure to the ambient atmosphere. PAC treatment introduces nitrogen and carbon irrespective of the substrate used for the upper or lower plate, as evident from the increased intensity N1s and C1s peaks in the XPS analysis (Figure 1A&B) and shown numerically in Tables 1 & 2.Table 1. Atomic percentage of oxygen (O), carbon (C) and nitrogen (N) on PDMS after different treatments. The error represented is the SEM.Table 2. Atomic percentage of oxygen (O), carbon (C) and nitrogen (N) on glass after different treatments. The error represented is the SEM.

[0165] To deduce more about the carbon species on the surfaces following the treatments described herein, the C1s peaks were deconvoluted at 4 different binding energies 284.2 eV, 286 eV, 287.5 eV and 289 eV associated with C-C, C-0 (or C-N), C=O (or C=N) and O-C=O respectively (Figure 1C). The C1s peak for the untreated (UT) surfaces is shown in Figure 1D. Qualitatively, the C1s peaks on two plate materials, PDMS and glass, were similar. PAC treatment of the upper plate and APPJ treatment introduced diverse functional groups on the respective surfaces like carbonyl (C=O) carboxylic groups (O-C=O) compared to O2 plasma treatment on the respective surfaces.

[0166] ESR spectra of PDMS after different treatments show characteristic peaks representing unpaired electrons on the surfaces treated with O2 plasma, PAC and APPJ (Figure 1E). This corroborated the presence of radicals on the treated surfaces. The spectra were similar for PDMS and quartz.

[0167] Hydrophobic recovery can occur for PDMS, which results in the properties, particularly the water contact angle, returning to the pre-treatment state. It occurs due to low molecular chains from the matrix migrating to the surface and / or the rotation of oxidized groups into the bulk so as to lower the surface energy. These processes are facilitated by the mobility of molecular chains and groups at and near the surface, hence, increased cross-linking of the treated surface layer improves the stability of the surface treatment. The effect of different treatments on the surfaces and their ageing profile was characterized using the wettability to water. The samples were stored at ambient conditions and room temperature. The time point of 0 denotes the measurement immediately after the treatment (within 10 minutes). Untreated PDMS has a water contact angle of 109° ± 2, which changed to 5° ± 2°, 28.0° ± 1.2° and 4° ± 2° respectively after O2 plasma, PAC and APPJ treatments (Figure 2A). PDMS treated with either O2 plasma or APPJ regained its original wettability in 4 days.

[0168] Untreated glass registered 62.37° ± 0.04°, which changed to 6° ± 2°, 26° ± 2° and 5° ± 3° respectively for O2 plasma, PAC and APPJ treatments (Figure 2B). None of the treatments retained native wettability on glass for up to 14 days. PAC, being a thin carbonaceous polymer film (estimated to be 7.5 ±1.1 nm using ellipsometry) did not retain native substrate wettability for up to 3 months on glass and PDMS with contact angle values of 71° ± 2° and 79° ± 5° respectively. In addition to exposure to air, the disintegration in surface properties is a thermally activated process. Hence, appropriate anaerobic storage conditions at colder temperatures can preserve the surface properties for longer durations.

[0169] Root mean square roughness (Rrms) value of untreated PDMS was 7.4 ± 1.2 nm and it did not change significantly after any of the treatments other than PAC as shown in Table 3. PAC-treated PDMS shows a wrinkled pattern with Rrms value of 91 ± 2 nm. PDMS being a flexible substrate undergoes thermal expansion and contraction at a rate different to the coating, thereby resulting in a characteristic wave-like pattern. The glass surface did not register any characteristic change in roughness after the treatments.Table 3: Rrms values (nm) of PDMS and glass surfaces before and after different treatments. The error represented is the SEM.Example 3: Covalent immobilisation of fibronectin

[0170] As covalent immobilization necessary for strong protein attachment to the surface and ensuring bioactive protein motifs are exposed to the cells, the propensity for plasma-treated surfaces to covalently immobilise the key matrix protein fibronectin was investigated. Both APPJ and PAC-treated surfaces retained a significant amount of protein on the surface (P < 0.05) compared to O2 plasma and untreated samples (Figure 3A, B).Example 4: Attachment and Proliferation of human coronary artery endothelial cells (HCAECs)

[0171] To assess the ability of these plasma-modified surfaces to support cell growth, the attachment and proliferation of human coronary artery endothelial cells (HCAECs) on various surfaces over time was measured. With glass and PDMS being used for the upper and lower plates of microfluidic devices as described herein, cell attachment on these substrates was investigated. Minimal cell attachment was observed on both materials, while tissue culture (TC) plastic strongly promoted cell attachment. Relatively low cell attachments were observed across all plasma-treated PDMS samples (Figure 4). Similarly, O2 plasma treatment did not promote cell attachment relative to untreated glass, but morphologically, HCAECs on O2 plasma-treated surfaces appeared less rounded, suggesting greater attachment to the underlying substrate. However, PAC and APPJ treatment significantly improved cell attachment on glass, with more cells attached to these surfaces relative to O2 plasma-treated glass (Figure 4).

[0172] To better understand the propensity for these surfaces to support cell growth, HCAEC proliferation was assessed after both 3 (Figure 5A) and 5 days (Figure 5B) on PDMS and glass plates. With minimal cells attaching to untreated glass and PDMS plates, these samples were not included in the proliferation studies. Similar trends wereobserved across days 3 and 5, and on all time points, TC plastic strongly supported cell proliferation. On PDMS, both O2 plasma and APPJ treatment failed to support cell growth, with few cells present on the surface, even after 5 days. In contrast, PAC treatment resulted in strong cell proliferation, outperforming O2 plasma and APPJ treatment, showing a comparable ability to support cell growth to TC plastic.

[0173] On glass plates, similar to the attachment assay, HCAECs proliferated to a greater extent than similarly treated PDMS plates. While O2 plasma-treated glass performed poorly compared to TC plastic, PAC-treated glass promoted cell proliferation to the greatest extent, performing better than O2 plasma-treated glass. Both PAC and APPJ-treated glass plates promoted cell proliferation to a greater extent than TC plastic. Negligible differences were observed between plasma-treated glass plates on Day 5, this was due to a near confluent-monolayer forming across the samples. These results suggest that PAC treatment effectively supports cell proliferation on both PDMS and glass, while APPJ treatment strongly promotes proliferation on glass.Example 5: Microfluidic devices

[0174] Based on these results and their applicability to microfluidics, a microfluidic device was developed using these plasma treatments. Both components - glass lower plate and PDMS upper plate - were treated with the relevant plasma methods and irreversibly bonded. HCAECs were cultured within the devices with continuous perfusion for 3 days. Minimal differences in cell count were observed between plasma treatments in uncoated devices. However, after coating with fibronectin, more cells were present on PAC (P < 0.001) and APPJ (P < 0.0001) devices (Figure 6A).

[0175] While more cells were present on APPJ and PAC devices after fibronectin coating than O2 plasma-treated devices (P < 0.01 and P < 0.0001 respectively), APPJ devices also contained more cells than PAC devices (P < 0.01). Despite fibronectin promoting cell proliferation within the microfluidics, oxygen plasma-treated devices with a fibronectin coating had a comparable cell count to uncoated PAC and APPJ devices (P > 0.05), highlighting the superiority of these plasma treatments for cell-based microfluidic systems.

[0176] The expression of CD31 , a key endothelial cell marker, was also quantified.Without coating with fibronectin, HCAECs on O2 plasma devices expressed significantly less CD31 than PAC (P < 0.01) devices. APPJ devices on average showed higherlevels of CD31 compared to O2 plasma devices but this difference was not statistically significant (P > 0.05). CD31 levels between PAC and APPJ devices was also determined to not be statistically significant (P > 0.05) (Figure 6B (Fig 6D)). After coating with fibronectin, CD31 levels increased on all three device types, with on average PAC and APPJ showing higher CD31 levels. However, no statistically significant differences were observed between the fibronectin coated treatments (P > 0.05). Similar to the cell proliferation trend, HCAECs in uncoated PAC and APPJ devices expressed a non-significant difference in the levels of CD31 to fibronectin- coated oxygen plasma devices (P > 0.05).

[0177] Additionally, endothelial nitric oxide synthase (eNOS) levels were quantified, as a key enzyme associated with endothelial cell function. HCAECs on coated and uncoated oxygen plasma and PAC devices expressed low levels of eNOS relative to APPJ devices (Figure 6C (Fig 6E)). However, the addition of fibronectin resulted in reduced eNOS levels in all devices, with it being statistically significant for APPJ-treated devices (P < 0.0001) but not PAC or O2 plasma-treated devices (P > 0.05).

[0178] These results highlight the superiority of PAC and APPJ treatments for microfluidic cell culture systems described in the present disclosure relative to conventional O2 plasma methods.

[0179] All cell culture analyses within the microfluidic device were performed on the internal surface of the glass lower plate. This is because, in both the O2 plasma and APPJ devices, the internal surface of the PDMS upper plate did not register any significant cell attachment and hence growth. Whereas the channel surface characterization of the PAC device was difficult because of its poor bond performance.Example 6: Mechanical testing

[0180] Despite the advantages in terms of robust immobilization of protein and superior cell growth offered by PAC, microfluidic devices having only PAC treated surfaces had poor bond strength between the upper and lower plates and failed the leak test, burst test and strength tests that were performed. APPJ devices on the other hand had the best results, sustaining all the tests and not needing any additional baking or ageing tests. The results of the tests are shown in Table 4.Table 4: Results of the bond strength analysis performed on devices fabricated with a PDMS upper plate and glass lower plate with the internal surfaces of the microfluidic channels treated with various plasma treatments. Both PDMS and glass had the same treatment. O2 plasma devices required an additional baking step to sustain microfluidic experiments as it anneals the bond between the glass and PDMS lower and upper plates.

[0181] In the case of tensile testing, for both O2 plasma and APPJ devices, the T- shaped structure came off once pulled to a certain tension. This corresponds to the breakage stress of the glue which binds the T-shaped structure to the device. This value was 314 ± 17 kPa for O2 plasma and 274 ± 20 kPa for APPJ devices (n=3). Hence, for O2 plasma, bond strength can be estimated to be > 314 ± 17 kPa; for APPJ, it can be estimated to be >274 ± 20 kPa. In the literature, the burst pressure of PDMS bonded to glass after O2 plasma was estimated to be 510 kPa (Bhattacharya, S., et al., Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength. Journal of Microelectromechanical Systems, 2005. 14(3): p. 590-597).Example 7: Hybrid Microfluidic devices

[0182] Based on the results obtained on durability and bioactivity, it was concluded that PAC treatment is the best for materials such as PDMS, and APPJ is the best for materials such as glass (summarised in Table 5). However, only APPJ treatment on the upper and lower plates formed a strong bond between the upper and lower plates stable enough for experiments involving high fluid flow or shear, as are required for many applications (eg, organ-on-a-chip).Table 5. Summary of the treatments for PDMS and glass categorized under different properties. X represents poor performance and O represents suitable performance.

[0183] However, by combining APPJ-treated glass with PAC-treated PDMS, a hybrid device was developed that overcame the bond strength issues with PAC-treated devices, while enabling covalent immobilization of proteins and superior cell growth to standard methods.

[0184] For a hybrid device, the bonding surface on the upper plate was masked so that only the open microfluidic channel surfaces were exposed. PAC treatment was performed as described previously. Following this, the mask was removed, and the surface of the upper plate was APPJ treated and bonded to an APPJ-treated lower plate (Figure 7A) forming the enclosed microfluidic channels. This hybrid device showed comparable bond strength to APPJ-based devices while ensuring the internal surfaces provided superior cell proliferation relative to standard methods.

[0185] The microfluidic device was coated immediately with fibronectin, with HCAECs cultured as normal for comparison with O2 plasma devices. In the hybrid plasma-treated devices, near complete endothelialisation was observed, though this was not observed in O2 plasma-treated devices, with few cells observed at the walls. This was confirmed by the quantified cell distribution on those specific internal surfaces (Figure 7B).

[0186] The hybrid device registered better cell growth on both the top internal surface (P <0.05) and the bottom internal surface (P < 0.01) of the microfluidic channels compared to the O2 plasma device, establishing the superiority of this method to impart bioactivity to an enclosed microfluidic channel. Using this, HCAECs were cultured within the hybrid device under coronary shear conditions (12.7 dynes / cm2) for 3 days, with cells forming a confluent, aligned monolayer on the lower plate (as is typical ofendothelial cells exposed to shear) with near-complete endothelialisation on the top internal surface of the microfluidic channels. While HCAECs were more aligned on the APPJ-treated plate relative to the PAC-treated upper plate, HCAECs on both internal surfaces of the microfluidic channels were significantly more aligned than the same internal surfaces in low shear devices (P < 0.0001) (Figure 7C). Taken together, the hybrid device enables superior endothelialisation of microfluidic chips under both standard and coronary shear conditions.Example 8: Hybrid Devices Robustly Immobilise other ECM Proteins and Antibodies Against a Strong SDS Wash

[0187] Other biomolecules, such as Type I Collagen and antibodies, were immobilised in the hybrid chip and standard O2 plasma chips and characterised for stability against surfactants by washing channels with SDS. After the wash, confocal images were taken, and the mean fluorescence intensity was compared against O2 Plasma devices. Post-SDS wash, over 80 times more collagen remained on the hybrid device on both the top PDMS and bottom glass surfaces compared to the O2 Plasma devices (P < 0.05), with minimal signal detectable on all O2 Plasma device surfaces (Figure 8).

[0188] This characterisation was then extended to an antibody where a conjugated secondary goat anti-rabbit IgG antibody was immobilised in the chips and washed with SDS (Figure 9). After SDS washing, minimal antibody was detected on PDMS and glass surfaces of O2 plasma devices. Relative to these devices, on the hybrid device, over 6-times more antibody was detected on glass and 24-times more on PDMS (Figure 9). However, this difference was not statistically significantExample 9: Hybrid device strongly immobilise antibodies against shear- induced detachment

[0189] Antibodies were immobilised in the hybrid chip, which underwent multiple washings with PBS-T, and the remaining biomolecules were quantified. Initially, conjugated secondary goat anti-rabbit IgG antibody was immobilised and subjected to shear forces through vigorous washes with PBS-T. The biomolecules retained on the surface after the wash were quantified (Figure 10A). Over 2-times more antibody was retained on the hybrid chip on both the top PDMS (P < 0.01) and the bottom glass surface (P < 0.05) compared to the O2 Plasma devices (Figure 10A).

[0190] Continuing the investigation with a different experiment, an unconjugated rabbit anti-human eNOS primary antibody was immobilised on the chips. After the incubation, the chips were washed with PBS-T and blocked using BSA. This was followed by incubation with a conjugated secondary goat anti-rabbit IgG antibody. After a gentle wash with PBS, the quantity of immobilised secondary antibody was quantified using a confocal microscope. On the hybrid devices, both PDMS (P < 0.05) and glass (P < 0.05) surfaces retained over 4-times more antibodies relative to O2 plasma (Figure 10B).

[0191] In the next assay, the stability of immobilised goat anti-rabbit IgG antibodies on the hybrid chip was compared with standard O2 Plasma devices and APTES devices under shear stress. Typically to chemically immobilize biomolecules onto surfaces, toxic reagents such as APTES are used which link the O2 Plasma-treated surface and the antibody molecule through a covalent bond. Initially, a similar quantity of antibodies was detected on all the devices, regardless of whether they had a PDMS or glass surface (Figure 11). After multiple washes with PBS-T, the intensity of antibodies steeply decreased on the O2 Plasma devices. The hybrid and APTES groups had relatively higher intensity of antibodies retained on both PDMS and glass surfaces. However, there were no statistically significant differences among the three groups on both PDMS and glass surfaces (Figure 11).Example 10: PAC on PDMS & Glass and APPJ on Glass Promote SMC Attachment

[0192] The study examined SMC attachment on various plasma-treated surfaces. Similar to the results observed with ECs (above), strong cell attachment was observed on APPJ, PAC-treated glass, and TC surfaces (Figure 12). Cell morphology on the PAC-treated surfaces of both glass and PDMS appeared more spread out and elongated. In contrast, cells on APPJ-treated surfaces exhibited a rounder appearance. The APPJ-treated glass significantly outperformed UT (P<0.0001), O2 Plasma (P <0.01), and PAC (P <0.05). All treatments displayed significantly better SMC attachment than the untreated (UT) surface (Figure 12). On PDMS, PAC treatment was superior to both APPJ (P <0.05), O2 Plasma (P <0.05), and UT (P <0.001), although attachment remained lower compared to TC (P <0.001) (Figure 12).Example 11 : Fibronectin on PAC-PDMS Promotes Proliferation, and Collagen Promotes Contractility in an in vitro Well Plate Culture

[0193] SMC proliferation, growth kinetics, and phenotype are among the most important characteristics to study for applications such as tissue engineering and disease modelling. Initially, SMCs were cultured on various plasma-treated glass and PDMS for five days, identifying the combination of PAC-PDMS and APPJ-glass as the most effective. However, the cultures did not reach confluence. Later, three biomolecules were immobilised on plasma-treated PDMS surfaces: TGF-p, collagen-l, and fibronectin. After seven days, the proliferation and expression of a contractile marker known as SM actin (or a-actin, now called SMA) was investigated.

[0194] Both surface treatment (P <0.0001) and biomolecule coating (P <0.001) significantly impacted SMC growth. Confocal micrographs show a confluent-like layer of cells on fibronectin and collagen-coated PAC-PDMS. Regardless of the biomolecule, PAC treatment notably enhanced cell growth compared to APPJ treatment (P <0.01) (Figure 13A). Cell quantification reveals, on average, a greater number of cells on fibronectin surfaces than on collagen surfaces on both TC and PAC-PDMS; however, this difference is statistically insignificant between those biomolecule groups (P = 0.994). This may also suggest that a confluent layer of cells is established. Minimal cell growth was observed on UT surfaces, regardless of the biomolecule coating (Figure 13A).

[0195] The expression of SMA was significantly influenced by the treatments and biomolecule coating. SMA levels on a per-cell basis were notably higher on PAC-treated surfaces compared to other surfaces (Figure 13B). SMCs on collagen and fibronectin- coated PAC-PDMS exhibited increased SMA expression compared to equivalent groups on APPJ (P < 0.01). On average, collagen appeared to enhance contractility more than fibronectin, although this difference was not statistically significant (Figure 13B).Example 12: Fibronectin on APPJ-glass Promotes Proliferation, and Collagen Promotes Contractility

[0196] Similar to PDMS, surface treatments and biomolecule coating greatly enhanced cell growth and SMA expression on glass surfaces (P<0.001) (Figure 14). Although cellgrowth was minimal, UT-glass surfaces are more favourable for cell growth compared to Untreated PDMS. The APPJ and TC surfaces exhibited significantly improved growth of SMCs in comparison to O2 Plasma (P<0.05), with fibronectin and collagen-coated groups showing the highest growth against comparable biomolecule groups on O2 Plasma-treated surfaces (P<0.001). SMCs grown on APPJ and TC surfaces achieved confluent-like cell growth, demonstrating no significant difference between them (Figure 14A).

[0197] SMA expression was also significantly enhanced by surface treatments (P < 0.0001) and biomolecules (P < 0.001) when compared to the UT group. Minimal expression was observed on O2 Plasma and UT surfaces. The collagen-coated APPJ exhibited the highest SMA expression, with no statistically significant difference compared to the fibronectin-coated APPJ, but a statistically significant improvement in relation to the TGF-p coated APPJ (P < 0.01) and the UT APPJ (P < 0.001) (Figure 14B).Example 13: Hybrid Device Immobilised with Fibronectin Promoted near Confluent SMC Growth Under Flow

[0198] SMCs were cultured under low shear flow (WSS = 0.007 dyn / cm2) in both standard O2 plasma devices and hybrid devices immobilised with biomolecules (Figure 15). After five days of culture, hybrid devices immobilised with matrix proteins, fibronectin, and Type I collagen promoted significantly greater cell growth than O2 Plasma devices (Figure 15A-B). Cells formed a confluent monolayer on the fibronectin- coated hybrid device, with significantly more cells than the fibronectin-treated O2 Plasma on both the top (PDMS) (P<0.001) (Figure 15A) and bottom (glass) surfaces (P <0.01) (Figure 15B). The fibronectin-coated group showed significantly better performance on the top surface of the hybrid device than the collagen-coated group (P <0.01) (Figure 15A). However, the difference was statistically insignificant on the bottom glass surfaces (Figure 15B). Similar observations were noted between the fibronectin and collagen groups in O2 Plasma devices, indicating a significant effect of fibronectin on cell growth. Interestingly, the TGF-p group performed poorly in both hybrid and O2 Plasma devices (Figure 15).Example 14: APPJ Treatment Helps in the Micropatterning of ECM Proteins in a Microfluidic Device

[0199] When culturing SMCs in 2D well plates, the observations showed no statistically significant difference between the influence of fibronectin and collagen on their proliferation and contractility on APPJ-treated glass and PAC-treated PDMS (Figures 13 and 14). In the microfluidic culture, a potent effect of fibronectin on the growth kinetics was observed, which led to the conclusion that fibronectin promotes proliferation (Figure 15). This implies that collagen-l should promote contractility compared to fibronectin. From both these models, it was deduced that the effect of a biomolecule on SMC characteristics would be more obvious at the early stages of their growth. This is because SMCs, when confluent, tend to become more contractile irrespective of the matrix protein, making interpretations difficult. Culturing SMCs on micropatterned protein surfaces can constrain their growth and elongation, making it easy to compare their growth kinetics and phenotypes.

[0200] FITC-conjugated fibronectin and collagen-l were patterned onto APPJ-treated glass using stamps with 20 pm rails and 40 pm gaps (Figure 16A). Confocal microscopy revealed well-defined patterns of both proteins (Figure 16B). An APPJ- treated PDMS device was bonded over the stamped region (Figure 16C). A simple leak test indicated no leakage from the channels, demonstrating irreversible bonding between the glass and PDMS, thereby enclosing the micropatterns within the channel.Example 15: Organ-on-a-chip using PC Membrane Allowed Proof-of- Concept Co-culture of a Confluent Layer of ECs and SMCs

[0201] Co-culture microfluidic models (organs-on-chips) typically feature various types of cells cultivated on each side of a thin, porous membrane; to emulate a blood vessel, SMCs and ECs are grown. These membranes also attempt to replicate the basement membrane, a form of ECM that delineates boundaries between tissues. The membrane structure is designed to prevent cell migration while permitting soluble signalling cues to pass through the pores and enabling direct cell-cell contact in localised areas, thereby facilitating cell-cell communication. Consequently, these models hold significant promise for investigating blood vessel characteristics, disease pathogenesis, and related drug discovery.

[0202] For the same reason, having multiple cell types makes these models inherently challenging, especially since SMCs and ECs exhibit different growth kinetics and have varying nutritional requirements. Additionally, in a human artery, ECs primarily encounter frictional forces from blood flow, while SMCs experience cyclic stretching due to pulsatile pressure. All these factors contribute to the difficulty of developing a biomimetic organ-on-a-chip.

[0203] The growth surface in this context is the membrane, and its chemical, physical, and mechanical properties are crucial for the success of the model (Figure 17A). A commercially available PC membrane with 8 pm track-etched pores was used. The PC membrane was treated on both sides with S-APPJ and placed in conformal contact with an F-APPJ-treated PDMS device that housed the lumen. Another PDMS device, also treated with F-APPJ, was aligned and bonded on top to create the dual-channel organ- on-a-chip (Figure 17A). As discussed in the previous examples, F-APPJ treatment was sufficient to facilitate bonding between PDMS and glass or PDMS to PDMS. Furthermore, the fast treatment requires only half a minute to complete, in contrast to the slower method, which takes six minutes. Therefore, opting for F-APPJ for bonding significantly reduced fabrication time.

[0204] A consideration here is that only the two PDMS adapters bond to each other due to the formation of Si-OH groups on them after the APPJ treatment. No covalent bond forms between the PC and PDMS surfaces. Therefore, the membrane was cut to size so that it barely covered the channel surface. This was to ensure increased contact between the two PDMS devices and to provide a larger bonding area. The PC membrane is also only 8.8 ± 1.2 pm thick, which aids in sandwiching it between the two PDMS adapters.

[0205] However, the absence of an irreversible bond between PDMS and the PC membrane presents specific challenges. Due to the membrane’s thin and hydrophilic nature, it becomes excessively wet during experiments. This causes the membrane to expand, leading to bending and deformation that distorts its planar arrangement. The lack of bonding exacerbates this transition, further reducing the membrane's integrity. Additionally, the non-bonded areas trap air bubbles, which migrate into the channels and obstruct the flow of media and the growth of cells. Despite these challenges, confluent-like EC and SMC layers were successfully grown on either side of the membrane (Figure 17B).

[0206] Also, PDMS adapters typically need to have a certain thickness to form a robust chip that can be handled appropriately during the experimental process. Unfortunately, the thickness of the chips poses a challenge during confocal imaging due to the limited working distance of microscope objectives. Hence, the chips were sliced on a vertical plane perpendicular to the membrane and imaged to visualise the two layers of cells. Due to how thin the membrane was, the layers could not be visualised distinctively. In addition to this, SEM imaging of the membrane by itself revealed its non-uniform pore distribution (Figure 17C). A few pores can be seen merged, forming significantly larger pores in many areas, indicated by arrows (Figure 17C). This could lead to cells and media of one type crossing the membrane to the other channel. Hence, results demonstrate that it is plausible to make multicell devices in which the cells are separated by membranes, allowing cell-cell communication and transfer of soluble factors through them.

Claims

CLAIMS1. A microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein a first portion of internal surface of the one or more enclosed microfluidic channels comprises a surface film having a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said surface film comprising at least carbon, nitrogen and oxygen; and wherein a second portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said second portion of internal surface being substantially free of nitrogen.

2. The microfluidic device according to claim 1, wherein the surface film has a thickness of less than about 20 nm, or from about 1 to about 10 nm.

3. The microfluidic device according to claim 1 or claim 2, wherein the surface film has an elemental nitrogen content from about 10 to about 30 atomic % as measured by XPS.

4. The microfluidic device according to any one of claims 1 to 3, wherein the surface film has an elemental oxygen content from about 10 to about 45 atomic % as measured by XPS.

5. The microfluidic device according to any one of claims 1 to 4, wherein the surface film has an elemental carbon content from about 10 to about 40 atomic % as measured by XPS.

6. The microfluidic device according to any one of claims 1 to 5, wherein the second portion of said internal surface has an elemental nitrogen content that is less than about 20 atomic %, or less than about 15 atomic %, or less than about 10 atomic %, or less than about 5 atomic %, or less than about 1 atomic % as measured by XPS.

7. The microfluidic device according to any one of claims 1 to 6, wherein the second portion of said internal surface has an elemental oxygen content from about 45 to about 85 atomic % as measured by XPS.

8. The microfluidic device according to any one of claims 1 to 7, wherein the second portion of said internal surface has an elemental carbon content from about 5 to about 35 atomic % as measured by XPS.

9. The microfluidic device according to any one of claims 1 to 8, wherein a majority of the internal surface of the one or more enclosed microfluidic channels comprises the surface film.

10. The microfluidic device according to any one of claims 1 to 9, wherein a minority of the internal surface of the one or more enclosed microfluidic channels is defined by the second portion of internal surface.

11. The microfluidic device according to any one of claims 1 to 10, wherein the surface film is a plasma activated coating (PAC).

12. The microfluidic device according to any one of claims 1 to 11, wherein the second portion of internal surface of the one or more enclosed microfluidic channels has been subjected to treatment by an atmospheric pressure plasma jet (APPJ).

13. A microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate; wherein at least one portion of internal surface of the one or more enclosed microfluidic channels has a modified elemental composition relative to the upper and the lower plate bulk elemental compositions, said at least one portion of internal surface being substantially free of nitrogen; wherein the at least one portion of internal surface has been subjected to treatment by an atmospheric pressure plasma jet (APPJ).

14. The microfluidic device according to any one of claims 1 to 13, wherein the upper plate and the lower plate independently comprise one or more of metal, glass, synthetic polymer, biopolymer, quartz, ceramic, hydrogels, metal oxides, and silicon.

15. The microfluidic device according to claim 14, wherein the glass comprises one or more of silicate glass, borosilicate glass, and soda-lime glass.

16. The microfluidic device according to claim 14, wherein the synthetic polymer comprises one or more of PDMS, PTFE, polycarbonate, polycaprolactone, polysulfone, polyurethane, polyethylene terephthalate, polyethylene, poly(methacrylate), polymethyl methacrylate, styrene-ethylene-butylene-styrene (SEBS), polylactic acid, polyglycolide, poly lactic-co-glycolic acid (PLGA), polystyrene, nitrocellulose, nitrocellulose blends, nitrocellulose-cellulose acetate, and cyclic olefin copolymer.

17. The microfluidic device according to claim 14, wherein the biopolymer comprises one or more of silk, alginate, collagen, gelatin, agarose, chitosan, dextran, polyhydroxyalkanoates (PHA), polylactic acid (PLA), starch and cellulose.

18. The microfluidic device according to claim 14, wherein the hydrogel comprises one or more of alginate, agarose, collagen, gelatin, gelatin methacrylate (GelMA), hyaluronic acid (HA), fibrin, polyacrylamide (PAA) and polyethylene glycol (PEG).

19. The microfluidic device according to any one of claims 1 to 18, wherein the upper plate and the lower plate are bonded together and the bond strength between the upper plate and the lower plate is, for example, greater than about 200 kPa.

20. The microfluidic device according to any one of claims 1 to 19, wherein the one or more enclosed microfluidic channels further comprise a membrane configured within the one or more enclosed microfluidic channels so as to define two channels separated by the membrane.

21. The microfluidic device according to claim 20, wherein a surface of the membrane has a modified elemental composition relative to the elemental composition of the bulk membrane.

22. The microfluidic device according to claim 20 or claim 21 , wherein the membrane comprises one or more of PDMS, polycarbonate, polyethylene, polyethylene terephthalate, polysulfone, polystyrene, parylene, polycaprolactone, polyethylene glycol diacrylate, polyurethane, polymethyl methacrylate, cyclin olefin copolymer, and polyamide.

23. The microfluidic device according to any one of claims 1 to 22, further comprising one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the surface film and / or second portion of internal surface.

24. The microfluidic device according to any one of claims 20 to 23, further comprising one or more organic small molecules, biomolecules, and / or cells covalently bonded to or immobilised on the membrane and / or membrane surface.

25. A method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of: a) providing an upper plate having one or more open microfluidic channels defined therein; b) depositing a plasma enhanced chemical vapour surface film, said surface film comprising at least carbon, nitrogen and oxygen, in the one or more open microfluidic channels of the upper plate; c) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; d) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and e) bonding the upper plate to the lower plate thereby producing the microfluidic device.

26. The method according to claim 25, wherein activating a surface of the upper plate and a surface of the lower plate in step c) comprises treatment with an atmospheric pressure plasma jet.

27. A method of fabricating a microfluidic device comprising an upper plate, a lower plate, and one or more enclosed microfluidic channels defined between the upper plate and the lower plate, comprising the steps of: a) providing an upper plate having one or more open microfluidic channels defined therein; b) activating a surface of the upper plate including the one or more open microfluidic channels and activating a surface of the lower plate; c) arranging the activated surface of the upper plate and the activated surface of the lower plate, thereby forming the one or more enclosed microfluidic channels between the upper plate and the lower plate; and d) bonding the upper plate to the lower plate, thereby producing the microfluidic device and wherein activating a surface of the upper plate and a surface of the lower plate in step b) comprises treatment with an atmospheric pressure plasma jet (APPJ).

28. A microfluidic device according to any one of claims 1 to 19, produced by the method according to any one of claims 25 to 27.

29. A method of covalently bonding or immobilising organic small molecules or biomolecules to the surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to claim 23, comprising: a) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels; and b) covalently attaching one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels.

30. A method of covalently bonding or immobilising cells to surface film and / or second portion of internal surface of one or more enclosed microfluidic channels of the microfluidic device according to claim 23, comprising: a) depositing cells in a cell culture medium and contacting said cell culture medium with one or more of the enclosed microfluidic channels; b) covalently attaching biomolecules from the cell culture medium to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules; or a) contacting a liquid comprising one or more organic small molecules or biomolecules with one or more of the enclosed microfluidic channels and covalently attaching said one or more of the organic small molecules or biomolecules to the surface film and / or second portion of internal surface of the one or more enclosed microfluidic channels; b) contacting a medium comprising cells with one or more of the enclosed microfluidic channels; and c) attaching the cells to the covalently attached biomolecules31. The microfluidic device according to claim 23 or claim 24, or the method according to claim 29 or claim 30, wherein the biomolecules comprise one or more of proteins, polypeptides, polysaccharides, polynucleotides, nucleotides, oligonucleotides, nucleic acids, enzymes, coenzymes, antibodies, drug molecules, hydrogels, antioxidants, growth factors, vitamins, lipids, glycosaminoglycans, and combination biomolecules thereof.

32. A microfluidic device according to any one of claims 1 to 24 for use in an organ- on-a-chip device, a lab-on-a-chip device, a separation device, a cell / biomarker- capture device, a diagnostic device, a synthesis device, or a sensor device.

33. The use of a microfluidic device according to any one of claims 1 to 24 in the manufacture of an organ-on-a-chip device, lab-on-a-chip device, a separationdevice, a cell / biomarker-capture device, a diagnostic device, a synthesis device, or a sensor device.

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