Microfluidic device with pillars

Abstract

The invention provides a microfluidic device (100) for the formation of lipid bilayers, wherein the microfluidic device (100) comprises a first fluid inlet (111), a first fluid outlet (112), and a first microfluidic channel (110) configured to fluidically connect the first fluid inlet (111) and the first fluid outlet (112), wherein: (A) the first microfluidic channel (110) comprises a membrane formation section (200) having a membrane formation section axis (Emfs) of elongation, wherein the channel wall (115) at the membrane formation section (200) comprises a membrane formation wall section (215); (B) the membrane formation section (200) comprises a plurality of micropillars (210); (C) the plurality of micropillars (210) are configured in an n*m array parallel to the membrane formation section axis (Emfs) of elongation; wherein n is selected from the range of ≥1 and m is selected from the range of ≥2; wherein the n*m array is configured to define (i) n+1 elongated subchannels (110a,110b,110c...) within the first microfluidic channel (110) and (ii) per row of the n rows m-1 fluidic connection sections (105); wherein each of the plurality of micropillars (210) tapers in directions parallel to the membrane formation section axis (Emfs) of elongation towards respective adjacent micropillars (210); (D) the membrane formation wall section (215) is configured in a wave-like shape parallel to the membrane formation section axis (Emfs) of elongation.

Description

[0001] Microfluidic device with pillars

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a microfluidic device, an application method using such a microfluidic device, and a system comprising such a microfluidic device. The invention further relates to a kit of parts comprising the microfluidic device.

[0004] BACKGROUND OF THE INVENTION

[0005] Microfluidic devices are known in the art. W02009069608A1, for instance, describes planar lipid-bilayer membrane arrays (1) using a microfluid having been preliminarily saturated with water by dipping in water, which comprise microchannels (2) wherein the planar lipid-bilayer membrane arrays (1) are connected to a liquid supply port and are arranged in parallel and microchambers (3) having openings in both sides of the microchannels (2).

[0006] EP3915545A1 describes a device, comprising: a first inlet microchannel configured to receive a first solution and provide a first stream comprising the first solution; a second inlet microchannel configured to receive a second solution and provide a second stream comprising the second solution; and a third microchannel configured to receive the first stream and the second stream, wherein the third microchannel has a first region adapted for flowing the first stream and the second stream and a second region adapted for mixing the first stream and the second stream to provide a third stream comprising a mixture of the first solution and the second solution, and wherein the second region of the third microchannel has a hydraulic diameter of 20 microns to 300 microns and a width of 200 microns to 300 microns.

[0007] SUMMARY OF THE INVENTION

[0008] Developing new drugs is a complex, time-consuming, and expensive process. Nearly half of all drug targets and key drug-metabolizing enzymes are found in intracellular environments, compartmentalized by semipermeable barriers, the cell membranes. Inspired by their lipidic bilayer structure, lipid-based biomimetic platforms are being developed to understand the biochemical and biophysical processes at the cellular membrane level.

[0009] The demand for adequate models mimicking membrane barriers also requires the development of screening protocols that provide reproducibility, standardization, throughput, and sustainability to support the drug discovery process. The prior art may, for instance, describe the use of in vitro models using living cells and in vivo animal studies. However, these models may be difficult to conduct, may require specific infrastructures and technical expertise, may present low reproducibility, and may be expensive and timeconsuming. Additionally, regarding a drug rational design, these systems may fail to support structure-related modification decisions, since they do not provide an understanding of drugmembrane interactions at a molecular level. Furthermore, the prior art may describe the use of lipid biomimetic systems with high-throughput screening (HTS) methods in multiple well plates to standardize the drug discovery process. However, while these HTS methods have been valuable in decreasing the attrition rate of pharmaceutical industries, they present limitations in processing time and require costly equipment.

[0010] Considering the pharmaceutical industry's demands for efficient in vitro high throughput screening tools, microfluidic technology emerges as a promising solution to address challenges in lipid biomimetic models for screening applications, presenting cost-effective platforms with improved reproducibility and throughput. Microfluidics involves the transformation of commonly used lipid-based biomimetic models, like supported lipid bilayers and lipid vesicles, to miniaturized approaches and their evolution into a new generation of bilayer models. These include pore-suspended and free-standing lipid bilayers, black lipid membranes, and droplet interface bilayers. However, such methods for assessing a lipid bilayer reaction to drugs may typically entail the use of micropipettes to transfer giant unilamellar vesicles (GUVs) that are mechanically or osmotically stretched into a chamber that contains a solution dissolving the drug of interest. These methods require trained technicians and large scale lab facilities to form GUVs using complex techniques and deliver them to another chamber containing drugs to treat the GUVs. Yet further, such methods do not provide good control over the concentrations of solution and the reversibility of the membrane treatment with drug.

[0011] Furthermore, microfluidic methods as described in the prior art may provide planar bilayer systems, which may often not be compatible with experimental techniques such as e.g. optical tweezers. The planar orientation of the bilayer may result in a relatively large annulus (or torus), which may be undesirable as the annulus may have a different refraction index which may (negatively) impact optical experiments.

[0012] Hence, it is an aspect of the invention to provide an alternative microfluidic device, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. According to a first aspect, the invention provides a microfluidic device for the formation of lipid bilayers. In embodiments, the microfluidic device may comprise a first fluid inlet, a first fluid outlet, and a first microfluidic channel configured to fluidically connect the first fluid inlet and the first fluid outlet. The first microfluidic channel may, in embodiments, comprise a channel wall defining a channel height (He). Additionally, in embodiments, the first microfluidic channel may comprise a membrane formation section having a membrane formation section axis (Emfs) of elongation. In embodiments, the channel wall at the membrane formation section may comprise a membrane formation wall section. Moreover, in embodiments, the membrane formation section may comprise a plurality of micropillars. In embodiments, each of the plurality of micropillars may be configured to extend along the full channel height (He). Furthermore, in embodiments, the plurality of micropillars may be configured in an n*m array parallel to the membrane formation section axis (Emfs) of elongation. In embodiments, n may be selected from the range of >1. Moreover, in embodiments, m may be selected from the range of >2. The n*m array may, in embodiments, be configured to define n+1 (parallelly configured) elongated subchannels within the first microfluidic channel. Additionally, in embodiments, the n*m array may be configured to define per row of the n rows m-1 fluidic connection sections. Especially, in embodiments, adjacent elongated subchannels may be configured fluidically connected at the m-1 fluidic connection sections configured between adjacent micropillars. Further, in embodiments, the plurality of micropillars each may have a maximum width (Wmax) defined in a direction perpendicular to the membrane formation section axis (Emfs) of elongation (and perpendicular to the channel height He). Yet further, in embodiments, each of the plurality of micropillars may taper from its maximum width (Wmax), at least in directions parallel to the membrane formation section axis (Emfs) of elongation, towards respective adjacent micropillars. Moreover, in embodiments, the membrane formation wall section may be configured in a (sinusoidal or triangular) wavelike shape parallel to the membrane formation section axis (Emfs) of elongation. In embodiments, a first distance (dl) may be defined between the membrane formation wall section at the maximum width (Wmax) of the micropillars and the membrane formation section axis (Emfs) of elongation. Conversely, in embodiments, a second distance (d2) may be defined between the membrane formation wall section at the fluidic connection sections between the micropillars and the membrane formation section axis (Emfs) of elongation. In embodiments, the first distances (dl) may be smaller than second distances (d2). Hence, in specific embodiments, the invention may provide a microfluidic device for the formation of lipid bilayers, wherein the microfluidic device may comprise a first fluid inlet, a first fluid outlet, and a first microfluidic channel configured to fluidically connect the first fluid inlet and the first fluid outlet, wherein: (A) the first microfluidic channel may comprise a channel wall defining a channel height (He); wherein the first microfluidic channel may comprise a membrane formation section having a membrane formation section axis (Emfs) of elongation, wherein the channel wall at the membrane formation section may comprise a membrane formation wall section; (B) the membrane formation section may comprise a plurality of micropillars, wherein each of the plurality of micropillars may be configured to extend along the (full) channel height (He); (C) the plurality of micropillars may be configured in an n*m array parallel to the membrane formation section axis (Emfs) of elongation; wherein n may be selected from the range of >1 and m may be selected from the range of >2; wherein the n*m array may be configured to define (i) n+1 elongated subchannels within the first microfluidic channel and (ii) per row of the n rows m-1 fluidic connection sections; wherein the plurality of micropillars may each have a maximum width (Wmax) defined in a direction perpendicular to the membrane formation section axis (Emfs) of elongation; and wherein each of the plurality of micropillars may taper from its maximum width (Wmax) at least in directions parallel to the membrane formation section axis (Emfs) of elongation towards respective adjacent micropillars; (D) the membrane formation wall section may be configured in a wave-like shape parallel to the membrane formation section axis (Emfs) of elongation, wherein first distances (dl) defined between the membrane formation wall section at the maximum width (Wmax) of the micropillars and the membrane formation section axis (Emfs) of elongation, may be smaller than second distances (d2) defined between the membrane formation wall section at the fluidic connection sections between the micropillars and the membrane formation section axis (Emfs) of elongation.

[0013] Such a microfluidic device provides the benefit of enabling users to conveniently form artificial cell membranes of various compositions in a high-throughput manner and perform drug screening tests on them. Hence, with such a microfluidic device it may be possible to test drugs on artificial cell membranes in-vitro in a relatively simple manner. No special skills may be required for application of the device. Using only two conventional syringe pumps and following a fast and straightforward protocol artificial cell membranes may be formed and may be tested on the herein described microfluidic device. In this way, the effect of drugs (e.g. antibiotics), proteins, toxins, microplastics, and any other type of reagents may be screened on cell membranes in-vitro in a robust and high-throughput manner.

[0014] The membrane formation section filled with several micropillars (especially combined with a bubble trap, see also further below) may provide high-throughput membrane formation, with several stable membranes forming per microchannel at a time. Using multiple membranes on a single device may further allow various experiments on different membranes without the time-consuming process of forming new membranes for each new experiment. Furthermore, the herein described embodiments may especially be suitable for mechanical characterization when a large number of experiments with different lipid compositions are usually required, in comparison to designs with a single membrane per microchannel which may have reproducibility and throughput issues.

[0015] All the steps of forming membranes, treating them with drug, performing measurements and characterizations, and doing (fluorescent) imaging may be done all on one (kit comprising the) microfluidic device described herein. A single microfluidic device may be applied to form and test relatively high numbers of membranes (e.g. up to 30 membranes) simultaneously (within less than 2 minutes), therewith decreasing costs and required time for high-throughput experiments.

[0016] Furthermore, the microfluidic device may provide the benefit of flexibility, as it may be compatible with various different tests and experiments that may need to be performed on membranes, such as e.g., fluorescent and confocal microscopy, optical tweezers, etc. The microfluidic device may especially be compatible with optical experiments such as optical tweezers due to a reduced annulus (and hence reduced optical interference by the refraction index of the annulus) presenting in the microfluidic device as described herein compared to existing microfluidic devices for membrane formation. Yet further, due to the material used to fabricate the microfluidic device, even harsh chemical may be used as solution on the device.

[0017] The invention may herein primarily be described in the context of drugmembrane interaction, such as in the context of the delivery of antibiotics. It will be clear to the person skilled in the art, however, that the invention is not limited to such embodiments. The method of the invention may, for instance, also be employed to delivery of proteins to the lipid bilayer to influence membrane stability and function.

[0018] The invention may thus provide a microfluidic device (or “device”) for the formation of lipid bilayers. In a further aspect, the invention may provide a use of the microfluidic device as described herein for (high-throughput) drug screening.

[0019] The term “microfluidic device” may herein refer to a device for the manipulation of small amounts of fluids using channels with sizes of ten to hundreds of micrometers. Microfluidic devices are known in the art. Amongst others, the invention provides a microfluidic device comprising a microfluidic channel with a plurality of micropillars, e.g. for (artificial) lipid bilayer formation. Especially, the invention provides a microfluidic chip. Furthermore, the term “lipid bilayers” may refer to a thin polar membrane made of two layers of lipid molecules. Hence, herein, also the term membrane may be applied. For example, the membrane may comprise two layers of amphiphilic phospholipids that have a hydrophilic phosphate head and a hydrophobic tail consisting of two fatty acid chains, wherein the hydrophobic tails of the two layers may be configured towards each other to form the membrane.

[0020] In embodiments, the microfluidic device may comprise a first fluid inlet and a first fluid outlet. The first fluid outlet may be configured downstream of the first fluid inlet. The terms “upstream” and “downstream” relate to an arrangement of items or features relative to the propagation of fluids from a fluid providing means (e.g. a pump or syringe) wherein relative to a first position within a flow of fluid from the fluid providing means, a second position in the flow of fluid closer to the fluid providing means is “upstream”, and a third position within the flow of fluid further away from the fluid providing means is “downstream”.

[0021] Further, the microfluidic device may comprise a first microfluidic channel configured to fluidically connect the first fluid inlet and the first fluid outlet. In embodiments, the microfluidic channel may thus comprise a hollow channel. Especially, the microfluidic channel may be configured to provide passage for a fluid through the microfluidic device. Hence, in embodiments, fluid may be provided to the microfluidic device at the first fluid inlet. During operation of the device, in embodiments, the fluid may flow in a direction from the first fluid inlet towards the first fluid outlet. The first fluid inlet and the first fluid outlet may, in embodiments, be configured in fluidic contact with each other. The term “fluidic contact” may especially indicate that a fluid may flow between the elements which are indicated to be in fluidic contact. Especially, herein, a gas (mixture, e.g. air) or a liquid, like an aqueous solution, may flow between the two elements.

[0022] The first microfluidic channel may, in embodiments, comprise an elongated channel, i.e., the channel may have one dimension (e.g. length) significantly larger than the other dimensions of the microfluidic channel (e.g. width, height, diameter). The (first) microfluidic channel may in embodiments have a microfluidic channel axis of elongation (or “microfluidic channel axis”). The microfluidic channel axis may in embodiments comprise any arbitrary shape. The microfluidic channel axis may in embodiments especially define a smooth line. The microfluidic channel axis may e.g. comprise a straight axis in embodiments. Yet, in alternative embodiments, the microfluidic channel axis may be curved or may, e.g., comprise one or more bends. The membrane formation section axis (Emfs) of elongation (or just “membrane formation section axis”) may in embodiments coincide with the first microfluidic channel axis (in the formation section).

[0023] Especially, the first microfluidic channel may comprise a (first) channel wall. In embodiments, the channel wall may comprise a smooth or rounded wall (e.g. when the channel comprises a cylinder). Alternatively, in embodiments, the channel wall may be faceted. Especially, in such embodiments, the channel wall may comprise three facets (such as e.g. a channel having a triangular cross-section), four facets (such as e.g. a channel having a rectangular cross-section), or more facets (such as e.g. having a polygonal cross-section). In embodiments, the channel wall may define a channel length (Lc) and a channel height (He). In embodiments, the microfluidic channel may approximate a cuboid shape. Hence, in embodiments, a cross-section of the microfluidic channel may approximate a square or rectangular shape. In such embodiments, the channel wall may define the channel height (He) and may additionally define a channel width (Wc). In embodiments, Lc>Hc, such as Lc>2*Hc, like Lc>3*Hc, especially Lc>5*Hc. Similarly, in embodiments, Lc>Wc, such as Lc>2*Wc, like Lc>3*Wc, especially Lc>5*Wc.

[0024] The channel height (He) is especially defined perpendicular to the channel width (Wc). The channel height (He) and the channel width (Wc) may in further embodiments define a plane perpendicular to the microfluidic channel axis (at any longitudinal position in the microfluidic channel).

[0025] The (local) channel dimension(s) may herein further be defined at a given longitudinal position in the channel.

[0026] The term “longitudinal”, in “a longitudinal position” (or “longitudinal location”) may herein especially refer to a (predetermined, given, and / or determinate) position or location along a length of an elongated object, such as along a length of the microfluidic channel, or e.g. along a length of a membrane formation section, bubble trap section (see also further below), etc. A longitudinal position in the microfluidic channel may for instance refer to a (predetermined or quantifiable) position, especially cross-sectional position, along the channel axis.

[0027] The channel wall of the microfluidic channel may, in embodiments, enclose a channel space. If it is described herein that a specific element is configured in the microfluidic channel this may, in embodiments, indicate that the specific element is configured in the channel space. The microfluidic channel may have a channel axis of elongation configured, especially perpendicular to the channel height (He) (and channel width (Wc)). It is note that the channel axis may comprise a straight axis, in embodiments. The channel axis may be configured substantially linear. Yet, in alternative embodiments, the channel axis may be curved, and / or may, e.g., comprise one or more bends. The channel axis may especially comprise any arbitrary shape. The channel axis may further be configured parallel to a shortest line connecting the fluid inlet to the fluid outlet via the microfluidic channel.

[0028] In alternative embodiments, the microfluidic channel may approximate a cylindrical shape. Hence, in embodiments, a cross-section of the microfluidic channel may approximate a circular shape. In such embodiments, the channel height (He) may essentially be a (cross-sectional) diameter of the microfluidic channel. Especially, in such embodiments, the channel height (He) may be an equivalent circular diameter (De) of a cross-section (perpendicular to a direction of elongation) of the microfluidic channel. The equivalent circular diameter (or ECD) (or “circular equivalent diameter”) of an (irregularly shaped) two- dimensional shape is the diameter of a circle of equivalent area. For instance, the equivalent circular diameter of a square with side a is 2*a*SQRT(l / 7t). For a circle, the diameter is the same as the equivalent circular diameter. Would a circle in an xy-plane with a diameter D be distorted to any other shape (in the xy-plane), without changing the area size, then the equivalent circular diameter of that shape would be D.

[0029] In yet alternative embodiments, the microfluidic channel may have any other elongated shape. For example, in embodiments, a cross-section of the microfluidic channel may approximate a triangular or polygonal shape. In such embodiments, the channel height (He) may be an equivalent circular diameter (De) of a cross-section (perpendicular to a direction of elongation) of the microfluidic channel.

[0030] The channel height (He) may, in embodiments, be selected from the range of >0.02 mm, such as from the range of >0.04 mm, like from the range of >0.06 mm, especially from the range of >0.08 mm. Further, in embodiments, the channel height (He) may be selected from the range of <1 mm, such as from the range of <0.5 mm, like from the range of <0.2 mm, especially from the range of <0.1 mm. Similarly, in embodiments, the (channel) equivalent circular diameter (De) may be selected from the range of >0.02 mm, such as from the range of >0.04 mm, like from the range of >0.06 mm, especially from the range of >0.08 mm. Further, in embodiments, the equivalent circular diameter (De) may be selected from the range of <1 mm, such as from the range of <0.5 mm, like from the range of <0.2 mm, especially from the range of <0.1 mm. Analogously, in embodiments, the (average) channel width (Wc) may be selected from the range of >0.5 mm, such as from the range of >1 mm, like from the range of >1.5 mm, especially from the range of >2 mm. Further, in embodiments, the (average) channel width (Wc) may be selected from the range of <30 mm, such as from the range of <20 mm, like from the range of <10 mm, especially from the range of <5 mm. Furthermore, in embodiments, the channel length (Lc) may be selected from the range of >20 mm, such as from the range of >30 mm, like from the range of >40 mm, especially from the range of >50 mm. Further, in embodiments, the channel length (Lc) may be selected from the range of <150 mm, such as from the range of <100 mm, like from the range of <80 mm, especially from the range of <60 mm.

[0031] Note that, in embodiments, the channel dimensions may not necessarily be constant over the whole channel, e.g., at one cross-section the channel width (Wc) may be larger than at a different cross-section. In other words, at one cross-section the channel may be narrower (or wider) than at a different cross-section. Hence, the herein described dimensions (Lc, He, Wc, and De) may refer to average dimensions. The channel dimensions may herein further be defined at a given longitudinal position in the channel.

[0032] The term “approximate” and its conjugations herein, such as in “to approximate a shape”, may refer to being nearly identical to, especially identical to, the following term, for example nearly identical to a circular cross-section or a semi-cylindrical shape. For example, a channel may define a cylindrical shape but for a defect. In particular, an object approximating a first shape may herein refer to: a first shape realization encompassing the object, wherein the first shape realization is defined as the smallest encompassing shape of the (2D or 3D, respectively) object wherein the first shape realization has the shape of the first shape, wherein a ratio of the area (volume) of the first shape realization to the area (volume) of the object may be < 1.2, especially < 1.1, such as <1.05, especially <1.02. For instance, a channel may approximate a semi-cylindrical shape, wherein the first shape realization may be defined as the smallest encompassing semi -cylindrical shape of the channel, wherein a ratio of the volume of the first shape realization to the volume of the channel is < 1.2, especially, especially < 1.1, such as <1.05, especially <1.02, including 1. Further, if the dimensions of the first shape are defined, the term approximate may refer to the object and the first shape being superimposable (in 2D or 3D, respectively) such that an intersection between the object and the first shape covers at least n% of the object and at least n% of the shape, wherein n is at least 90%, such as at least 95%, especially at least 98%, such as at least 99%, including 100%.

[0033] The microfluidic channel may further, in embodiments, comprise a membrane formation section. The membrane formation section may have a membrane formation section axis (Emfs) of elongation. In embodiments, the membrane formation section may be configured such that during operation of the microfluidic device (by providing fluids for membrane formation to the device) membranes (or lipid bilayers) may be formed in the membrane formation section in a direction parallel to the membrane formation section axis (Emfs) of elongation. Therefore, in embodiments, the membrane formation section may comprise a plurality of micropillars.

[0034] Herein, micropillars may refer to structures configured in the microfluidic channel (especially in the membrane formation section) and having a shape such that, in embodiments, the micropillars may have one dimension equal to one dimension of the microfluidic channel. Especially, in embodiments, (at least part of) the micropillars may have a micropillar height (HM) (equal to or) that may extend along the (full) channel height (He). More especially, in embodiments, each of the plurality of micropillars may be configured to extend along the full channel height (He). Or alternatively, such as in embodiment indicated above, (at least part of) the micropillars may have a micropillar height (HM) (equal to or) that may extend along the full equivalent circular diameter (De). In other words, the micropillar height (HM) may be at least 95%, such as at least 98%, like at least 99%, especially at least 99.5%, including 100% of the channel height (He). Especially, in embodiments, the micropillars may each be configured in the membrane formation section such that in one dimension of the microfluidic channel (especially the channel height (He)) there may not be empty space (or an opening) between the channel wall and the micropillar. Conversely, in such embodiments, the micropillars may each be configured in the membrane formation section such that in the other dimensions of the microfluidic channel (especially the channel length (Lc) and the channel width (Wc)) there may yet be empty space (especially an opening) between the channel wall and the micropillar.

[0035] As indicated above, in embodiments, the microfluidic channel may comprise a channel wall. In embodiments, at the membrane formation section the channel wall may comprise a membrane formation wall section. Especially, in embodiments, the membrane formation wall section may refer to the section of the channel wall defined along the subchannel length (Lsc) and configured parallel to the micropillar height (HM).

[0036] In some embodiments, the microfluidic channel may have a rectangular (or polygonal) cross-sectional shape. In such embodiments, the channel wall may comprise a channel bottom section, a channel top section, and a spacer wall configured to connect the channel bottom section to the channel top section. Hence, in such embodiments, the spacer wall may define the channel height (He), especially, the channel height (He) may be defined between the channel bottom section and the channel top section. Furthermore, in such embodiments, (where the microfluidic channel may have a rectangular (or polygonal) cross-sectional shape) the membrane formation wall section may refer to the spacer wall defined along the subchannel length (Lsc).

[0037] Further, in embodiments, the membrane formation section may comprise a plurality of micropillars. Especially, in embodiments, the membrane formation section may comprise at least 2 micropillars (configured in a longitudinal position relative to each other in the membrane formation section), like at least 3, such as at least 4, like at least 6, especially at least 12 micropillars. Further, in embodiments, the membrane formation section may comprise at most 80 micropillars, such as at most 60, like at most 40, especially at most 30 micropillars. Especially, in embodiments, the plurality of micropillars may be configured in an array, such as an n*m array, parallel to the membrane formation section axis (Emfs) of elongation. In specific embodiments, the plurality of micropillars may be configured in an n*m array, wherein the n rows of micropillars may be configured parallel to the membrane formation section axis (Emfs) of elongation, such that the m columns may extend along the direction of elongation.

[0038] Herein, in embodiments, n may be selected from the range of >1, such as from the range of >2, like from the range of >3. Selecting n>2 may be advantageous as this may enable the formation and analysis of dual-stacked lipid bilayers within the microfluidic device. Especially, in embodiments, n may be selected from the range of <10, such as from the range of <8, like from the range of <6. Conversely, in embodiments, m may be selected from the range of >2, such as from the range of >3, like from the range of >4. Especially, in embodiments, m may be selected from the range of >5, such as from the range of >8, like from the range of >10. Furthermore, in embodiments, m may be selected from the range of <25, such as from the range of <20, like from the range of <15. For example, in embodiments, n=l and m=2 (i.e. there may be 2 micropillars). In an alternative example, in embodiments, n>2 and m>3, e.g. n=2 and m=3 (i.e. there may be 6 micropillars). Such embodiments may be beneficial as 6 micropillars may allow formation of up to 4 lipid bilayers, therewith enabling triplicate experiments in one single use of the microfluidic device. In a yet alternative example, in embodiments, n=2 and m=15 (i.e. there may be 30 micropillars).

[0039] In embodiments, the n*m array (of micropillars) may be configured to define n+1 (parallelly configured) elongated subchannels within the first microfluidic channels. Hence, in embodiments, the n rows of micropillars may divide the (first) microfluidic channel (at least in the membrane formation section) into n+1 elongated subchannels. In embodiments, the n+1 elongated subchannels may have a subchannel length (Lsc) defined as the largest distance between the micropillar configured closest to the first fluid inlet and the micropillar of the same row m configured closest to the fluid outlet. Especially, in embodiments, the subchannel length (Lsc) may be selected from the range of 0.5-30 mm, such as from the range of 2-25 mm, like from the range of 5-20 mm, especially from the range of 8-15 mm. The subchannel length (Lsc) especially depends on (i) the number of columns (i.e. the value of m) of the n*m array, (ii) a micropillar length (LM, see also further below), and a membrane formation distance (dm, see also further below).

[0040] For example, in embodiments where n=l there may be n+l=2 elongated subchannels. In such embodiments, the micropillars may be configured centered, i.e., such that the microfluidic channel may be split into two elongated subchannels equal in size. However, this may not necessarily be the case.

[0041] In a further example, in embodiments where n=2 there may be n+l=3 elongated subchannels. In such embodiments, the elongated subchannels may effectively comprise two wall-adjacent subchannels, i.e., configured adjacent to the membrane formation wall section, and one central subchannel, i.e., configured between the wall-adjacent subchannels (and may thus in itself not be configured directly adjacent to the membrane formation wall section).

[0042] Furthermore, in embodiments, the n*m array (of micropillars) may be configured to define per row of the n rows m-1 fluidic connection sections. Hence, in embodiments, between each of the m columns of micropillars an opening (such as a through hole) may exist, where fluid from the adjacent elongated subchannels may be fluidically connected. In other words, in embodiments, fluidic connections sections may be configured between micropillars of a row, such that the micropillars and the fluidic connection sections may alternate, i.e., provide an alternating pattern. Hence, in embodiments, adjacent elongated subchannels may be configured fluidically connected at the m-1 fluidic connection sections configured between adjacent micropillars. Note that, in embodiments, the fluidic connection sections of adjacent subchannels may thus coincide, such that said subchannels may be fluidically connected.

[0043] In embodiments, the membrane formation section may thus comprise micropillars configured adjacent to each other in a direction perpendicular to the direction of elongation (i.e. n rows of micropillars), such that the membrane formation section may comprise n+1 subchannels. Furthermore, the membrane formation section may thus comprise micropillars configured adjacent to each other in a direction parallel to the direction of elongation (i.e. m columns of micropillars), such that the membrane formation section may comprise m-1 fluidic connection sections.

[0044] In embodiments, the plurality of micropillars may each have a maximum width (Wmax) defined in a direction perpendicular to the membrane formation section axis (Emfs) of elongation (and perpendicular to the channel height (He)). Especially, in embodiments, the maximum width (Wmax) may be selected from the range of 25-500 pm, like from the range of 50-300 pm, such as from the range of 50-250 pm, especially from the range of 75-250 pm. Further, in embodiments, the maximum width (Wmax) may be selected from the range of 75- 200 pm, like from the range of 100-150 pm.

[0045] Further, in embodiments, the plurality of micropillars may each have a micropillar length (LM) defined parallel to the membrane formation section axis (Emfs) of elongation (and perpendicular to the channel height (He)). Especially, in embodiments, the micropillar length (LM) may be selected from the range of 25-2000 pm, like from the range of 50-1000 pm, such as from the range of 50-750 pm, especially from the range of 75-500 pm. Further, in embodiments, the micropillar length (LM) may be selected from the range of 100- 500 pm, like from the range of 200-400 pm.

[0046] In embodiments, at least part of, especially each of, the micropillars may have a width that may vary along its micropillar length (LM). Especially, in embodiments, at least part of, especially each of, the plurality of micropillars may taper from its maximum width (Wmax) along its micropillar length (LM). More especially, in embodiments, at least part of, especially each of, the plurality of micropillars may taper from its maximum width (Wmax) at least in directions parallel to the membrane formation section axis (Emfs) of elongation towards respective adjacent micropillars. As a result, in embodiments, a cross-section of the micropillars perpendicular to the micropillar height (HM) may have a shape with a varying width, such as e.g. a shape selected from the group comprising a (semi-)circular shape, an oval shape, an ovoid shape, an obovoid shape, a diamond (or rhombus or parallelogram) shape, a triangular shape, a kite shape, a trapezium shape, and a polygonal shape. In specific embodiments, one or more of the plurality of micropillars may have a diamond-like cross- sectional shape (defined in a plane perpendicular to the micropillar height (HM). Such embodiments may be beneficial as the tapering (or narrowing) corners of the diamond shapes of adjacent micropillars may allow fluids from adjacent subchannels to gently collide at the fluidic connection section between said adjacent tapering micropillars.

[0047] As described above, the subchannel length (Lsc) especially depends on (i) the number of columns (i.e. the value of m) of the n*m array, (ii) the micropillar length (Lm), and a membrane formation distance (dm). Especially, in embodiments, Lsc=m*Lm+(m-l)*dm. In embodiments, the micropillars may be configured at a distance from each other, such that the fluidic connection sections may be configured between the micropillars. In embodiments, during operation, lipid monolayers from the subchannels may join at the fluidic connection sections, therewith forming lipid bilayers (or membranes) at the fluidic connection sections. Therefore, in embodiments, the distance between the micropillars may be referred to as a membrane formation distance (“or a membrane length”) (dm). In embodiments, the membrane formation distance (dm) may be selected from the range of 25-750 pm, like from the range of 25-500 pm, such as from the range of 50-500 pm, especially from the range of 75-250 pm. Further, in embodiments, the membrane formation distance (dm) may be selected from the range of 50-250 pm, like from the range of 100-200 pm.

[0048] In exemplary embodiments, the first microfluidic channel may comprise 2*3 micropillars having a micropillar length (Lm) of 500 pm and being configured at a distance (i.e. the membrane formation distance (dm) from each other of 150 pm. In such embodiments, the subchannel length (Lsc) may be 3*500 + (3-1)* 150 pm = 1800 pm. In an alternative example, in embodiments, the first microfluidic channel may comprise 2*15 micropillars having a micropillar length (Lm) of 250 pm and being configured at a distance (i.e. the membrane formation distance (dm) from each other of 150 pm. In such embodiments, the subchannel length (Lsc) may be 15*250 + (15-1)* 150 pm = 5850 pm.

[0049] In general lipid bilayers may be relatively fragile structures that are easily influenced by environmental changes, such as e.g. pressure, and temperature of fluid flows. Hence, in embodiments, the micropillars may be configured to guide fluids through the membrane formation section in a more gentle and controlled fashion, therewith enabling formation of the lipid bilayers. To further stabilize environmental factors during the formation of the lipid bilayers, in embodiments, the membrane formation wall section may be configured in a streamlined shape parallel to the membrane formation section axis (Emfs) of elongation. In embodiments, a first distance (dl) may be defined between the membrane formation wall section at the maximum width (Wmax) of the micropillars and the membrane formation section axis (Emfs) of elongation. Conversely, in embodiments, a second distance (d2) may be defined between the membrane formation wall section at the fluidic connection sections and the membrane formation section axis (Emfs) of elongation. Hence, the first distance (dl) and the second distance (d2) may both be axis-wall distances, i.e., distances between the non-linear membrane formation section wall and the membrane formation section axis (Emfs) of elongation. In embodiments, the membrane formation wall section may be configured such that dl<d2. Especially, in embodiments, the membrane formation wall section may be configured such that the first distances may be smaller than the second distances, i.e., dl<d2. Such embodiments may thus be achieved by configuring the membrane formation wall section in a stream-lined shape. Especially, in embodiments, the membrane formation wall section may be configured in a wave-like shape. Thus, the membrane formation wall section may especially be configured in a shape or outline having successive curves. Hence, in embodiments, the membrane formation wall section may not be configured in a straight line, i.e., may not be linear. Especially, the membrane formation wall section may be configured to approximate the shape of a wave. For example, in embodiments, the wave-like shape of the membrane formation wall section may comprise a sinusoidal wave. Further, in embodiments, the wave-like shape of the membrane formation wall section may comprise a triangular wave. In specific embodiments, the membrane formation wall section may be configured in a wave-like shape that follows the (same but mirrored or opposite) pattern that a fluidic flow (especially the flow comprising the aqueous phase-organic phase interface) experiences at the n*m array (especially at the micropillars). In embodiments, the micropillars may be configured having an expanding (up to Wmax) and contracting (from Wmax) geometry in a longitudinal direction, i.e., in the direction of the flow. In such embodiments, the membrane formation wall section may be configured to mimic that same (but mirrored or opposite) pattern, such that the fluidic flow (especially the flow comprising the aqueous phase-organic phase interface) experiences the same expanding and contracting geometry (and may therewith experience a more constant or controlled pressure level).

[0050] Furthermore, a third distance (d3) may be defined between the membrane formation wall section at the maximum width (Wmax) of the micropillars and the micropillars of an adjacent row (i.e. a wall-adjacent row) m of the n*m array at the maximum width (Wmax) of the respective micropillars. Conversely, a fourth distance (d4) may be defined between the membrane formation wall section and the fluidic connection sections between the micropillars of that row m. In embodiments where the membrane formation wall section may be essentially linear, the first distance (dl) may essentially be equal to both the second distance (d2) and the fourth distance (d4). However, for improving the streamlined flow of fluids through the channel it may be desired that the first distance (dl) may be smaller than the second distance (d2) (and the third distance (d3) may be smaller than the fourth distance (d4). Especially, in embodiments, dl<0.98*d2, such as dl<0.95*d2, like dl<0.90*d2. Similarly, in embodiments, d3<0.98*d4, such as d3<0.95*d4, like d3<0.90*d4. In embodiments, the first distance (dl) may be selected from the range of 50-1500 pm, such as from the range of 100-1000 pm, like from the range of 200-800 pm. Further, in embodiments, the second distance (d2) may be selected from the range of 50-2500 pm, such as from the range of 150-2000 pm, like from the range of 250-1000 pm. Yet further, in embodiments, the third distance (d3) may be selected from the range of 50-1500 pm, such as from the range of 100-1000 pm, like from the range of 200-800 pm. Yet further, in embodiments, the fourth distance (d4) may be selected from the range of 50-2500 pm, such as from the range of 150-2000 gm, like from the range of 250-1000 gm.

[0051] In specific embodiments, the membrane formation wall section may be configured in a (sinusoidal or triangular) wave-like shape mirroring the tapering of the plurality of micropillars (in a row m adjacent to the wall). Hence, in such embodiments, the peaks and valleys of the wave may essentially fully align with a wave-like shape that may exist due to (i) the tapering of the micropillars and (ii) the alternating pattern of the micropillars and the fluidic connection sections. As a result, in embodiments, the subchannels created by the n*m array and the membrane formation wall section may sequentially contract (or converge) and expand (or diverge) along the direction of elongation of the microchannel. Such embodiments may be beneficial (especially in embodiments where n>2) as the shape of the membrane formation wall section may help relieve pressure differences between wall-adjacent subchannels and central subchannels, therewith streamlining the flow of fluid through the microchannel. A more streamlined and consistent flow (velocity) of fluid through the (subchannels of the) microchannel may improve membrane formation efficiency. With the herein described configuration that may follow the fluid-flow pattern (contracting - expanding width of walls in the channel), the pressure and the flow velocity on the interface of each lipid monolayer during and after membrane formation may be equalized, therewith enabling smooth zipping of the lipid monolayers into lipid bilayers, and insuring flatness and improved long-term stability of the membrane under ongoing flow. Hence, the embodiments described herein may provide a symmetric (i.e. balanced fluidic pressure at the fluidic connection section) flow, which may be ideal for membrane formation.

[0052] In embodiments, the microfluidic device may comprise a material selected from the group comprising a glass material and a polymeric material. Especially, in embodiments, the microfluidic device may comprise a polymeric material, such as e.g. selected from the group comprising: polydimethylsiloxane (PDMS), a curable optical adhesive (NOA), and epoxybased negative photoresist such as SU8 polymer. Further, in embodiments, the microfluidic device may comprise a glass material, such as e.g. a quartz (i.e. silica sand), a limestone, a dolomite, borosilicate, glass-ceramics, and recycled glass. The curable optical adhesive may especially comprise 50-70 (wt)% of a Mercapto ester and 30-50 (wt)% Triallyl Isocyanurate. For example, the curable optical adhesive may comprise a combination of Pentaerythritol tetrakis(3-mercaptopropionate) with Triallyl Isocyanurate. Moreover, the curable optical adhesive may e.g. comprise Norland Optical Adhesive (NOA), especially Norland Optical Adhesive 81. However, it may be clear to the skilled person that other optical adhesives may work as well.

[0053] The microfluidic device may, in embodiments, consist of essentially one type of material, i.e., the microfluidic device may be monolithic. Alternatively, in embodiments, the microfluidic device may comprise a combination of different materials as herein described. For example, the microfluidic device may comprise both polydimethylsiloxane (PDMS) and a curable optical adhesive (such as e.g. Norland Optical Adhesive (NOA)). In another example, the microfluidic device may comprise both borosilicate and Norland Optical Adhesive (NOA). Especially, in embodiments, the spacer wall as described above may for example comprise a cured optical adhesive, whereas the channel bottom section and / or the channel top section may comprise a glass, such as borosilicate.

[0054] In embodiments, the above described structure of the microfluidic device may be provided using a 3D printing technique, such as one or more of the group comprising: two- photon polymerization (2PP), digital light processing (DLP), stereolithography (SLA), fused deposition modelling (FDM), selective laser sintering (SLS), polyjet printing, and inkjet printing.

[0055] Further, in embodiments, the microfluidic device may comprise a light- transmissive material. Especially, in embodiments, the microfluidic device may essentially consist of light-transmissive materials. Such embodiments may be beneficial as the microfluidic device may be applicable in light-based experiments such as e.g. microscopy (confocal, optical tweezers, fluorescent imaging). Further, in embodiments, the microfluidic device may comprise a material that may have a high resistance to common solvents and reagents (e.g. chloroform, hexane, methanol, acetone and aqueous buffers) used for lipid bilayer formation. Such embodiments may be beneficial as the microfluidic device may be applicable for providing a variety of lipid bilayers in a wide range of experimental set-ups.

[0056] In specific embodiments, the microfluidic device may comprise a cured optical adhesive comprising 50-70 (wt) % of a Mercapto ester and 30-50 (wt)% Triallyl Isocyanurate, such as cured Norland Optical Adhesive 81 (or N0A81). Such embodiments may be beneficial as cured Norland Optical Adhesive 81 may absorb small amounts of chloroform (commonly used as organic solvent for lipid membrane formation), therewith reducing the effect of the annulus in the microfluidic device and improving the device efficiency. The annulus, also known as torus, may refer to a reservoir of lipid and solvent molecules trapped between the two layers of the lipid bilayer (in other words between leaflets of the membrane) at a connection point between the formed lipid bilayer (configured at the fluidic connection section) and an adjacent micropillar. In embodiments, if the annulus is significantly large, experiments such as e.g. interfacing optical tweezers on chip with the membranes may be impaired. Furthermore, cured Norland Optical Adhesive 81 may be relatively resistant to various solvents and reagents and light-transmissive.

[0057] Furthermore, in embodiments, the microfluidic device may have a height (H) and a width (W) defined in a plane perpendicular to the membrane formation section axis (Emfs) of elongation. Especially, in embodiments, the height (H) may be selected from the range of 0.02-50 mm, such as from the range of 0.05-30 mm, like from the range of 0.1-25 mm. More especially, in embodiments, the height (H) may be selected from the range of 0.02-24 mm, such as from the range of 0.03-15 mm, like from the range of 0.04-10 mm. Moreover, in embodiments, the microfluidic device may have a width (W) defined perpendicular to the membrane formation section axis (Emfs) of elongation and perpendicular to the height (H). Especially, in embodiments, the width (W) may be selected from the range of 0.05-50 cm, such as from the range of 0.1-30 cm, like from the range of 0.2-20 cm, especially from the range of 0.3-10 cm. More especially, in embodiments, the width (W) may be selected from the range of 0.05-25 cm, such as from the range of 0.1-15 cm, like from the range of 0.2-10 cm, especially from the range of 0.2-5 cm. Further, in embodiments, the microfluidic device may have a length (L) defined parallel to the membrane formation section axis (Emfs) of elongation. Especially, in embodiments, the length (L) may be selected from the range of 1-50 cm, such as from the range of 2-30 cm, like from the range of 3-20 cm, especially from the range of 5-10 cm. More especially, in embodiments, the length (L) may be selected from the range of 1-35 cm, such as from the range of 2-25 cm, like from the range of 3-8 cm.

[0058] In specific embodiments, the microfluidic device may comprise a microfluidic chip. Hence, in such embodiments, the microfluidic device may have a relatively flat shape. Especially, in embodiments, L>2*H, such as L>3*H, like L>5*H. More especially, in embodiments, L<20*H, such as L<10*H. Further, in embodiments, W>2*H, such as W>3*H, like W>5*H. Yet further, in embodiments, W<20*H, such as W<10*H. In specific embodiments, the microfluidic device may be elongated (or longer than it is wide), i.e., L>W. However, this may not necessarily be the case. Hence, in embodiments, L>W.

[0059] The microfluidic device may, in embodiments, be wider than the (first) microfluidic channel. As mentioned above, (artificial) lipid bilayers may be relatively sensitive or fragile structures. The first microfluidic channel may be used to provide the lipid bilayers, however, for e.g. drug delivery experiments on the lipid bilayers, it may be desirable (or even necessary) to provide different fluids to the microfluidic device. Changing the input on the first fluid inlet may cause disturbances to the membrane formation section and may potentially damage the lipid bilayers. Therefore, it may be desired to provide the different fluid (containing active compounds, such as drug or protein, to be delivered to the membranes) via a less invasive route. Therefore, in embodiments, the microfluidic device may further comprise a second fluid inlet, a second fluid outlet, and a second microfluidic channel. In embodiments, the second microfluidic channel may be configured at least partially parallel to the first microfluidic channel. Furthermore, in embodiments, the first microfluidic channel may be configured physically separated from the second microfluidic channel. The first microfluidic channel and the second microfluidic channel may in embodiments be fluidically coupled, for instance via one or more connection channels, see further below.

[0060] The second microfluidic channel may be configured to fluidically connect the second fluid inlet and the second fluid outlet. In embodiments, the second microfluidic channel may thus comprise a hollow channel. Especially, the second microfluidic channel may be configured to provide passage for a fluid through the microfluidic device. Hence, in embodiments, fluid may be provided to the microfluidic device at the second fluid inlet. During operation of the device, in embodiments, the fluid may flow in a direction from the second fluid inlet towards the second fluid outlet. The second fluid inlet and the second fluid outlet may, in embodiments, be configured in fluidic contact with each other.

[0061] Hence, in specific embodiments, the microfluidic device may further comprise a second fluid inlet, a second fluid outlet, a second microfluidic channel configured to fluidically connect the second fluid inlet and the second fluid outlet, and one or more connection channels (see also further below), wherein the one or more connection channels may be configured to fluidically connect the second microfluidic channel to the first microfluidic channel. Such embodiments may provide the advantage that, after membrane formation using the first microfluidic channel, the second microfluidic channel may be used to provide active compounds (such as drugs or proteins) to the fragile lipid bilayers with limited disturbance of the lipid bilayers. Hence, with such embodiments, the risk of damaging the lipid bilayers during (high-throughput) screening experiments (e.g. drug delivery experiments) may be reduced. The second microfluidic channel may thus function for the introduction of a substitute media such as e.g. a second aqueous phase (see also further below) to the formed membranes, and may therefore also be referred to as replacement channel.

[0062] The second microfluidic channel may, in embodiments, comprise an elongated channel, i.e., the channel may have one dimension (e.g. length) significantly larger than the other dimensions of the microfluidic channel (e.g. width, height, diameter). Especially, the second microfluidic channel may comprise a (second) channel wall. The second microfluidic channel may have dimensions as described in relation to the first microfluidic channel, see above.

[0063] Note that, in embodiments, dimensions of the second channel may not necessarily be constant over the whole channel, .e.g., at one cross-section the second channel width may be larger than at a different cross-section. In other words, at one cross-section the second channel may be narrower (or wider) than at a different cross-section. Hence, the herein described dimensions (Lc, He, Wc, and De) may refer to average dimensions.

[0064] Furthermore, as opposed to the above described first microfluidic channel, in embodiments, the second microfluidic channel may not necessarily comprise a membrane formation section. However, in embodiments, the second microfluidic channel may yet comprise a membrane formation section as described herein.

[0065] In embodiments, the second microfluidic channel may be fluidically connected to the first microfluidic channel. Therefore, in embodiments, the microfluidic device may further comprise one or more connection channels (or “resistance channels”) configured to fluidically connect the second microfluidic channel to the first microfluidic channel. Therefore, in embodiments, each connection channel may have (i) a first joint configured where the respective connection channel is connected to (or joined with) the first microfluidic channel, and (ii) a second joint configured where the respective connection channel is connected to (or joined with) the second microfluidic channel.

[0066] In embodiments, the connection channels may function as a (flow) resistance, such that the flow of fluids from one microfluidic channel to the other microfluidic channel may more controllable. In other words, the connection channels may be configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from one microfluidic channel to the other microfluidic channel. Especially, in embodiments, the connection channels may be configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from one of the first microfluidic channel and the second microfluidic channel to the other one of the first microfluidic channel and the second microfluidic channel. Therefore, the one or more connection channels may be relatively long and narrow (compared to the first and / or second microfluidic channel). Each connection channel may have a (connection) channel axis (CA) defined between the first joint and the second joint as defined above. In embodiments, the one or more connection channels may each have a total channel length (LRC) defined along a (connection) channel axis (CA), i.e., defined between the first joint and the second joint. Especially, in embodiments, for each connection channel, the total channel length (LRC) may be selected from the range of 0.5-50 mm, such as from the range of 1-25 mm, like from the range of 2-15 mm. Additionally or alternatively, in embodiments, for each connection channel, the total channel length (LRC) may be selected from the range of 2-75 mm, like from the range of 2-50 mm, such as from the range of 3-40 mm, like from the range of 5-30 mm.

[0067] In further embodiments, the one or more connection channels may each have an equivalent cross-sectional circular diameter (DRC) defined perpendicular to the (connection) channel axis (CA) of the respective connection channel. Especially, in embodiments, the equivalent cross-sectional circular diameter (DRC) may be selected from the range of 25-1000 pm, such as from the range of 250-500 pm, like from the range of 75-300 pm, especially from the range of 100-250 pm. In specific embodiments, the equivalent cross-sectional circular diameter (DRC) may be selected from the range of 50-500 pm. Furthermore, in embodiments, the equivalent cross-sectional circular diameter (DRC) may be smaller (or narrower) than the equivalent circular diameter (De) of the (first and / or second) microfluidic channel. Especially, in embodiments, DRC<DC, such as DRC<0.90*DC, like DRC<0.8*DC, especially DRC<0.6DC.

[0068] In embodiments, the microfluidic device may comprise one connection channel. Alternatively, in embodiments, the microfluidic device may comprise two connection channels. Yet, in embodiments, the microfluidic device may comprise a plurality of connection channels, such as e.g. three or four. Especially, in embodiments, the one or more connection channels may comprise a first connection channel and a second connection channel. In embodiments, the first connection channel may be configured upstream of the membrane formation section and downstream of the first and second fluid inlets (and downstream of a bubble trap (section), see also further below). Conversely, in embodiments, the second connection channel may be configured downstream of the membrane formation section and upstream of the second fluid inlet. Hence, in embodiments, the one or more connection channels may comprise a first connection channel and a second connection channel; wherein the first connection channel may be configured upstream of the membrane formation section; and wherein the second connection channel may be configured downstream of the membrane formation section. Such embodiments provide the advantage that, should one of the resistance channels become clogged due to accumulation of e.g. lipids, active compounds or impurities, the other resistance channel may yet provide the resistance and improved controllability of the flow of fluids through the microfluidic device. The second connection channel, located downstream of the membrane formation section, may thus be incorporated in the design to provide a fallback position in case the first connection channel fails. Failure of the first connection channel may especially happen if the first connection channel is clogged by droplets formed via unwanted mixing happening between organic solvent (e.g. chloroform) and aqueous solution at the joints of the connection channels with the first microfluidic channel or even in the connection channel itself.

[0069] The term “connection channel” may in embodiments refer to a plurality of (different) connection channels.

[0070] The connection channel may, in embodiments, comprise linear (or essentially straight) channels. Alternatively, in embodiments, the connection channels may comprise curved channels. In specific embodiments, at least one of the one or more connection channels may meander. Therefore, in embodiments, the (connection) channel axis (CA) may also be curved or may also meander. Especially, in specific embodiments, at least one of the one or more connection channels may be configured in a meandering pattern. More especially, in embodiments, at least one of the one or more connection channels may be configured in a zigzag pattern. Such embodiments may be beneficial as a curved, meandering, or zigzag pattern may allow for larger length of the channel without drastically increasing the size of the microfluidic device. Hence, such embodiments provide relatively good resistance of the fluid flow, while maintaining a compact and manageable microfluidic device.

[0071] In order to further improve the flow of fluids through the microfluidic device, the microfluidic device may further, in embodiments, comprise a (first) bubble trap section. In embodiments, the (first) bubble trap section may be configured downstream of the first fluid inlet and upstream of the membrane formation section. Unwanted bubbles are commonly formed in microchannels at the interface between aqueous phase fluids and organic phase fluids. Such bubbles may disrupt the contact process between monolayers of lipids, therewith reducing lipid bilayer formation efficiency. The bubbles may have several possible origins: air trapping due to the geometry and various components of the system (tubing, connection, inlet, microchannels), wettability properties of the device material, temperature variations, and injecting both an organic and an inorganic solution into the device. Embodiments of microfluidic device may provide the benefit of applying a passive in-plane bubble trap to capture unwanted bubbles from the fluid flow and reduce their impact on the lipid bilayer formation. The herein described bubble trap may especially provide a novel passive bubble trap with an in-plane configuration and designed with an asymmetric architecture. Especially, the bubbly trap may ensure stable and robust screening experiments on the membranes.

[0072] In embodiments, the first microfluidic channel may comprise a bubble trap section having a bubble trap section axis (EBT) of elongation. In further embodiments, the channel wall at the bubble trap section may comprise a bubble trap wall section, extending along the channel height (He) (in embodiments, along a total of the channel height (He)). The bubble trap wall section is, in embodiments, configured in the first microfluidic channel. In further embodiments, the bubble trap section comprises a divider wall arrangement. The divider wall arrangement may, in embodiments, be configured to divide the first microfluidic channel in the bubble trap section in a first passage and a second passage. The first passage and the second passage may be configured parallel to each other (i.e. especially not (substantially) downstream or upstream from each other). In further embodiments, the bubble trap wall section and the divider wall arrangement define a primary first passage flow-through area (An) and a secondary first passage flow-through area (An) (both) from (or especially in) the first passage, especially wherein the primary first passage flow-through area (An) is configured upstream of the secondary first passage flow-through area (An). Further, in embodiments, the bubble trap wall section and the divider wall arrangement may (further) define a primary second passage flow-through area (A21) and a secondary second passage flow-through area (A22) (both) from (or especially in) the second passage. In further embodiments, especially wherein the primary second passage flow-through area (A21) is configured upstream of the secondary second passage flow-through area (A22). In specific embodiments, An>A2i and An <A22.

[0073] Hence, in specific embodiments, the first microfluidic channel may comprise a bubble trap section having a bubble trap section axis (EBT) of elongation, wherein the channel wall at the bubble trap section may comprise a bubble trap wall section extending along the channel height (He), wherein the bubble trap section my comprise a divider wall arrangement; wherein the divider wall arrangement may be configured to divide the first microfluidic channel in the bubble trap section in a first passage and a second passage; wherein the bubble trap wall section and the divider wall arrangement may define (i) a primary first passage flow-through area (An) and a secondary first passage flow-through area (A12) (both) from the first passage, wherein the primary first passage flow-through area (An) may be configured upstream of the secondary first passage flow-through area (A12), and (ii) a primary second passage flow- through area (A21) and a secondary second passage flow-through area (A22) (both) from the second passage, wherein the primary second passage flow-through area (A21) may be configured upstream of the secondary second passage flow-through area (A22); wherein A11AA21 and A12AA22.

[0074] Such microfluidic device may be used for a variety of microfluidic applications, such as especially membrane formation, requiring a bubble-free liquid flow. The device may comprise a bubble trap that may independently operate. Moreover, such bubble trap may be integrated in the microfluidic device allowing a direct and easy integration in a microfluidic system. The microfluidic device, especially the bubble trap, may be small compared to prior art systems. The microfluidic device may, in embodiments, be integrated in a chip. The microfluidic device may further allow relatively high liquid flows through the microfluidic device while still trapping the bubbles. Furthermore, the microfluidic device may be used for aqueous liquid flows as well as for organic liquid flows, multi-phase flows, and combinations of these flows. Furthermore, embodiments of the microfluidic device may be configured for tolerating harsh materials such as chloroform or acetone.

[0075] The herein described bubble trap may provide a novel passive bubble trap with an in-plane configuration and designed with an asymmetric architecture. Embodiments of the microfluidic device may provide the benefit of applying a passive in-plane bubble trap to capture unwanted bubbles from the fluid flow and to reduce their impact on a successive microfluidic application (such as membrane formation) in the microfluidic device. The passive in-plane configuration makes the device easy-to-fabricate. In embodiments, the bubbly trap may ensure stable and robust microfluidic applications in the device. The microfluidic device may further, in embodiments, be made of a transparent material allowing to use optical techniques to check the trapping of bubbles and / or for studying specific applications in the microfluidic device.

[0076] Herein the bubble trap section may function as a bubble trap. Therefore, also the term “bubble trap” may, in embodiments, be used referring to the bubble trap section. The invention may thus provide a microfluidic device (or “device”) comprising a bubble trap. In a further aspect, the invention may provide a method for trapping bubbles in a microfluidic device, especially the microfluidic device of the invention.

[0077] Amongst others, the invention provides a microfluidic device comprising a microfluidic channel with a bubble trap section, e.g. for trapping bubbles from a liquid flow to provide a bubble-free liquid flow. The bubble free liquid flow may successively be used in a microfluidic application (such as the above describe membrane formation) configured in the same microfluidic device, or e.g. in a further microfluidic device. The invention may, in embodiments, provide a microfluidic chip.

[0078] The terms “bubble trap”, “trapping bubbles”, and comparable terms especially refer to separating a gaseous phase from a liquid (flow). The gaseous phase may be present as visible bubbles. The gaseous phase may also be present as minute bubbles that optionally may coalesce forming larger bubbles. The bubbles are especially separated / extracted from the (liquid) flow such that downstream of the bubble trap, the liquid may be bubble-free. The bubble may, in embodiments, be contained in the bubble trap after trapping the bubble. Bubble traps are known in the art, and may for instance also be called “debubblers” or “degassers”.

[0079] The microfluidic channel is, in embodiments, especially configured for guiding a fluid flow from an upstream end (especially the first fluid inlet) of the microfluidic channel to a downstream end of the microfluidic channel (especially the first fluid outlet).

[0080] The term “microfluidic channel” may, in embodiments, refer to a plurality of (the same or different) microfluidic channels, e.g. to a first microfluidic channel and / or a second microfluidic channel, see also below. In embodiments, each microfluidic channel may be configured to fluidically connect an individual fluid inlet and a respective fluid outlet. For clarity reasons, herein, the fluid channel and the respective inlet and outlet may be indicated with comparable adjectives. For instance, a first microfluidic channel may fluidically connect a first fluid inlet and a first fluid outlet, a second microfluidic channel may fluidically connect a second fluid outlet and a second fluid outlet, and a further microfluidic channel may connect a further fluid inlet and a further fluid outlet. Hence, if the microfluidic channel is indicated as a “certain” microfluidic channel, this may imply that the (respective) fluid inlet and fluid outlet connected to (or “of’) the “certain” fluid channel may also be indicated as the “certain” fluid inlet and the “certain” fluid outlet, respectively.

[0081] Moreover if a specific element or section is configured in the certain microfluidic channel, also the specific element or section may be indicated as “certain” specific element, or “certain” section, herein for clarity reasons. For instance if a bubble trap section is configured in a first microfluidic channel, then this bubble trap may also be indicated as a “first” bubble trap section. Likewise, if a bubble trap section is configured in a second microfluidic section, this bubble trap section may also be indicated as a “second” bubble trap section. Yet, in embodiments, it may also be described that the first microfluidic channel comprises a bubble trap section and the second microfluidic channel comprises a bubble trap section. This may also be understood by the skilled person such that both (the first and the second) microfluidic channels may comprise a bubble trap section (wherein the bubble trap sections may be configured alike or different in embodiments).

[0082] The term “fluid” may, in specific embodiments, especially refer to a liquid (a liquid containing gas bubbles and / or a bubble-free liquid). In further specific embodiments, the term “fluid” may (also) refer to a gaseous fluid.

[0083] The microfluidic channel may thus in embodiments comprise the bubble trap section. In embodiments, the bubble trap section may comprise the divider wall arrangement. The divider wall arrangement may, in embodiments, be configured parallel to the bubble trap section axis (EBT). The divider wall arrangement may, in further embodiments, at least partly extend along the (full) channel height (He). The divider wall arrangement may, in embodiments, have a (divider wall) height (HSD), especially approximately equal to the channel height (He). In embodiments, there may be no opening or empty space between the divider wall arrangement and the channel wall in a direction parallel to the height of the divider wall arrangement. Yet, in further embodiments, there may be a very small opening between the divider wall arrangement and the channel wall in a direction parallel to the height of the divider wall arrangement. Such very small opening may especially have a size of no more than 120 pm to prevent a bubble to pass through the opening. The divider wall arrangement height (HSD) (and / or a height of subdivider elements, see further below) (especially at a given longitudinal position) may be at least 90%, especially at least 95%, like at least 99% (and especially no more than 100%) of the channel height (He) (at the given longitudinal position).

[0084] The divider wall arrangement may comprise a plurality of sections or “subdivider elements” defining the divider wall arrangement, especially defining openings between the subdivider elements (see further below). It will be understood that the “height” of the divider wall arrangement at these openings may be less than the divider wall arrangement height (HSD), and may especially be zero. The divider wall arrangement may further divide the microfluidic channel at the bubble trap section in the first passage and the second passage, see also further below. Hence, the divider wall arrangement may be configured such that in a longitudinal direction and in a direction perpendicular to the channel axis, there is empty space or openings between the divider wall arrangement and the channel wall.

[0085] As indicate above, at the bubble trap section, the channel wall may comprise a bubble trap wall section. Herein, the term “bubble trap wall section” may especially refer to a section of the channel wall defined along the bubble trap section axis (EBT) of elongation and configured parallel to the divider wall arrangement height (HSD). The term “bubble trap wall section” may, in embodiments, refer to a portion of the channel wall at the bubble trap section. It is noted that, in embodiments, a total length of the divider wall arrangement may be equal to or smaller than a total length of the bubble trap wall section. Further, especially, the bubble trap wall section may, in embodiments, comprise two distinct portions of the channel wall configured at opposite sides of the divider wall arrangement. The term “bubble trap wall section” may refer to two bubble trap wall sections, each one being defined by one of the two distinct portions of the channel wall. Herein, these two wall sections or two distinct portions of the channel wall, especially of the bubble trap wall section, may also be referred to as “two opposite wall portions of the bubble trap wall section”, see also further below. The bubble trap wall section may, in further embodiments, define the width of the microfluidic channel at a given longitudinal position (in the bubble trap section). Likewise, a height of the bubble trap wall section (at a given longitudinal position) may, in embodiments, (especially comprising a rectangular cross-section of the microfluidic channel) correspond to channel height (He) (at the given longitudinal position).

[0086] The divider wall arrangement and the bubble trap wall section may further, in embodiments, define the first passage and the second passage. Herein, the first passage and the second passage together may also be referred to as “the (two) passages”. In embodiments, the first passage may thus be defined by one of the two opposite wall portions (of the bubble trap wall section) and (“in combination with”) the divider wall arrangement, and especially the second passage may be defined by the other one of the two opposite wall portions and the divider wall arrangement.

[0087] The two passages may, in embodiments, be configured to provide a flow profile in the passages for allowing to trap any bubble (present in the fluid) in the bubble trap section. Based on the configuration of the bubble trap section, e.g., in embodiments, a bubble in the flow when entering the bubble trap section may flow into the second passage, whereas the liquid (part of the flow) may flow into the first passage. This different behavior (of the liquid phase and the gas phase) may be the result of a difference between (a size of) the primary first passage flow-through area An and (a size of) the primary second passage flow-through area A21. The effect may especially be observed for A21 / A11 being smaller than 1. The bubble may further be trapped (caught) in the bubble trap section, especially based on the difference between (a size of) the secondary first passage flow-through area A 12 and (a size of) the secondary second passage flow-through area A22 (arranged further downstream in the passages), especially wherein A12 / A22 is smaller than 1.

[0088] Hence, in embodiments, a primary ratio A21 / A11 is (configured to be) smaller than 1, such as smaller than 0.99. The primary ratio A21 / A11 may, in embodiments, be equal to or smaller than 0.95, such as 0.9 at maximum, like 0.8 at maximum. The primary ratio A21 / A11 may further be at least 0.05, such as at least 0.1, especially at least 0.3. In embodiment the primary ratio A21 / A11 may be selected from the range of 0.1-0.9, such as from the range of 0.3- 0.8. The primary ratio A21 / A11 may for instance be around 0.5, e.g. 0.5 ± 20%. Based on the primary ratio, a bubble entering the bubble trap may, in embodiments, be facilitated to flow into the first passage (and especially not in the second passage).

[0089] A secondary ratio of A12 / A22 may, in further embodiments, have a value that is described above in relation to the primary ratio A21 / A11. The secondary ratio of An ^ is, in further embodiments, selected from the range of 0.1-0.9, such as from the range of 0.3-0.8. The (value of the) secondary ratio may especially be selected independently from (the value of) the primary ratio. Yet, in specific embodiments, the primary ratio and the secondary ratio may have the same value.

[0090] In embodiments, A21 is configured at a primary second longitudinal position (of the microfluidic channel in the bubble trap section) and An is configured at a primary first longitudinal position (of the microfluidic channel in the bubble trap section). In specific embodiments, the primary second longitudinal position may be configured further upstream (especially closer to the (first and / or second) fluid inlet) then the primary first longitudinal position. Further, in embodiments, An and A21 are configured at the same (primary) longitudinal position in the microfluidic channel. A (minimal) distance (along the microfluidic channel axis) between the fluid inlet and A21 may, in specific embodiments, be equal to or less than the (minimal) distance between the fluid inlet and An. Having A21 configured at least not further away from the fluid inlet then An may further support guiding the bubble into the first passage, in embodiments.

[0091] In further embodiments, A22 is configured at a secondary second longitudinal position (of the microfluidic channel in the bubble trap section) and A12 is configured at a secondary first longitudinal position (of the microfluidic channel in the bubble trap section). In specific embodiments, the secondary second longitudinal position may be configured further upstream (especially closer to the fluid inlet) then the secondary first longitudinal position. In alternative embodiments, the secondary second longitudinal position may be configured further downstream then the secondary first longitudinal position. In yet further embodiments, A12 and A22 may be configured at a same longitudinal (respective) position in the microfluidic channel. The secondary second longitudinal position and the secondary first longitudinal position may be the same (- secondary - longitudinal position).

[0092] In further embodiments, the flow-through area between the primary first passage flow-through area and the secondary first passage flow-through area may vary (i.e. the flow- through area may be increased or be decreased in the first passage as a function of a longitudinal position in the first passage). As a result of a flow-through area that initially (especially downstream from the primary first passage flow-through area) increases and successively (further downstream) decreases in a downstream direction (especially at locations upstream from the secondary first flow-through area), a bubble may be trapped, especially kept, or maintained in the bubble trap section, especially in the first passage of the bubble trap section. Likewise, the flow-through area between the primary second passage flow- through area and the secondary second passage flow-through area may vary, in embodiments. As a result of a flow-through area that initially (especially downstream from the primary second passage flow-through area) increases and successively decreases in a downstream direction (especially upstream from the secondary second flow-through area), a bubble may be trapped especially kept (or maintained), in the bubble trap section, in embodiments. Moreover, in embodiments, a relatively large bubble may be split at the divider wall arrangement into (a bubble comprising) a first portion entering the first passage and (a bubble comprising) a second (especially smaller) portion entering the second passage, and especially the second portion may be trapped in the second passage of the bubble trap section.

[0093] In embodiments, a change in microfluidic channel width (Wc) as a function of the longitudinal position in the bubble trap, may result in the varying flow-through area in the first and / or second passage.

[0094] Hence, in embodiments, the bubble trap wall section and the divider wall arrangement further define a largest intermediate first passage flow-through area (A19) from the first passage configured between the primary first passage flow-through area (An) and the secondary first passage flow-through area (An). In embodiments, A19 >An and A19 > A12. In further embodiments, the bubble trap wall section and the divider wall arrangement further define a largest intermediate second passage flow-through area (A29) from the second passage configured between the primary second passage flow-through area (A21) and the secondary second passage flow-through area (A22). In further embodiments, A29 >A2i and A29 > A22.

[0095] Further, especially, a first intermediate ratio A19 / A11 may be selected from the range of 1.2-10, especially from the range of 1.5-5. Likewise, in embodiments, a second intermediate ratio A29 / A21 may be selected from the range of 1.2-10, especially from the range of 1.5-5. Based on such configuration, bubble trapping in the bubble trap section may further be assisted.

[0096] In further specific embodiments, A19 / A29 > 1. The ratio A19 / A29 may, in embodiments, be at least two, such as at least five. The ratio A19 / A29 may, in further embodiments, be ten at maximum, such as eight at maximum, and, in embodiments, five at maximum. Embodiments having a relatively large first intermediate ratio A19 / A11 may especially keep larger bubbles in the bubble trap relative to embodiments having a smaller first intermediate ratio (especially for embodiments wherein a total length of the divider wall arrangement is about the same). Furthermore, having A19 larger than A29 may further facilitate keeping the bubble in the bubble trap. If A19 is much smaller than A29 this may result in a relatively high pressure drop for the flow going through the first passage which may result in embodiments in pushing out a bubble caught in that passage, e.g., via one or more of the gaps in embodiment comprising subdivider elements, see below).

[0097] In further embodiments, a flow-through area may (gradually) reduce in size from the upstream end of one or more of the (first and second) passages to a longitudinal position comprising the respective primary (first or second) passage flow-through area. Additionally, or alternatively, the flow-through area may (gradually) increase in size (again) (starting) from the longitudinal position comprising one or more of the secondary (first and second) passage flow- through areas to the downstream end of the respective primary (first or second) passage.

[0098] In embodiments, the width of the channel in the bubble trap section may vary in in the longitudinal direction (a direction parallel to divider wall arrangement). Moreover, in further specific embodiments, a shortest distance (d7) in the bubble trap section between two opposite wall portions of the bubble trap wall section (especially arranged at opposite sides of the divider wall arrangement) is reduced in a direction from the fluid inlet (up) to a first longitudinal position, and the shortest distance (d7) (in the bubble trap section) (especially between the two opposite wall portions) is increased in a direction from a second longitudinal position to the fluid outlet. Further, especially, the wherein the first longitudinal position may comprise one or more of the primary first passage flow-through area An and the primary second passage flow-through area A21. In yet further embodiments, the second longitudinal position may comprise one or more of the secondary first passage flow-through area A12 and the secondary second passage flow-through area A22.

[0099] In further embodiments, the divider wall arrangement may be defined by a plurality (at least two) of subdivider wall elements separated by (a) gap(s) (configured between the subdivider elements). The divider wall arrangement may, in embodiments, e.g. comprise an array of k subdivider elements, and especially an array of at least two subdivider elements. In further embodiments, the subdivider elements may especially be configured parallel to the bubble trap section axis EBT). The subdivider elements may mutually be separated by k-1 gaps between the subdivider elements. The divider wall arrangement may in embodiment comprise at least two subdivider elements, such as at least three, or at least four, or in embodiments at least six subdivider elements. In embodiments k>3, such as k> 4, like k>6. In further embodiments k< 20, especially k<10, like k< 6, The number k may in embodiments, e.g. be selected from the range of 2-10, like from the range of 2-6.

[0100] In specific embodiments, the subdivider elements may especially be configured (in a line) parallel to (or coinciding with) the bubble trap section axis (EBT). The gap(s) may be configured for allowing liquid to flow between the first and second passage via the gap. The gap may further especially be configured such that a gas bubble may especially not pass through the gaps. A size of the gap (or “gap size”), especially a shortest distance between adjacently configured subdivider element is therefore, in embodiments, configured to be 150 pm at maximum, like 120 pm at maximum. The gap size is, in further embodiments, especially at least 10 pm, such as at least 25 pm, like at least 50 pm, and, in embodiments, at least 75 pm. Especially, each gap defines a fluidic connection between the first passage and the second passage. The gap size between a first pair of adjacently configured. The gap size between subdivider elements may be configured independently from another gap size between other subdivider elements (or configured in another bubble trap section in the microfluidic device). The fluid connection(s) may facilitate liquid to overtake (pass) the bubble in the bubble trap section.

[0101] In specific embodiments, the divider wall arrangement comprises a fragmented divider wall comprising an array of k subdivider elements configured parallel to the bubble trap section axis (EBT) of elongation, wherein the subdivider elements are mutually separated by k- 1 gaps between the subdivider elements; wherein k>2; especially wherein a shortest distance (d5) between adjacently configured subdivider elements is selected from the range of 50-120 pm.

[0102] In embodiments, any one of the subdivider elements may have a subdivider length (LSD) defined parallel to the bubble trap section axis (EBT) of elongation (and perpendicular to the channel height (He)). In embodiments, the subdivider length (LSD) may be selected from the range of 100-3000 pm, like from the range of 500-3000 pm, such as from the range of 1000-2500 pm, especially from the range of 1500-2000 pm. Further, in embodiments, the subdivider length (LSD) may be selected from the range of 1000-5000 pm, such as from the range of 1500-3000 pm. A ratio of the channel width (Wc) at a given longitudinal position (in the bubble trap section) to the subdivider length LLD) at the given longitudinal position may, in embodiments, be selected from the range of 0.1-5, like from the range of 0.25-3.

[0103] In further embodiments, the subdivider elements may comprise a tapered shape, tapering in a direction parallel to the bubble trap section axis (EBT). In specific embodiments, adjacently configured subdivider elements may taper in a direction towards each other. Tapering may positively guide a liquid flow from one of the passages to the other passage. Tapering may further prevent a bubble in the bubble trap to break-up. Tapering at the upstream end of the divider wall arrangement may, in further embodiments, allow increasing the flow rate without pinning the liquid phase at edges of the divider wall arrangement. The liquid phase preferably is eased to flow through the bubble trap, allowing it to overtake any bubble in the bubble trap.

[0104] Hence, in embodiments, adjacently configured subdivider elements have a tapering shape tapering in a direction towards each other. In alternative embodiments, one or more of the subdivider elements have a rounded shape or a blunt shape at their extremes facing adjacently configured subdivider elements.

[0105] The subdivider element may have a subdivider width (d6) (perpendicular to the subdivider length (LSD)) that may thus vary along the subdivider length (LSD). The subdivider element may, in embodiments, especially comprise a (maximum) subdivider width (d6) configured perpendicular to the channel height (He) (or divider wall arrangement height) and to the subdivider length (LSD). The (maximum) subdivider width (d6) may, in embodiments, be selected from the range of 50-500 pm, such as from the range of 50-400 pm, especially from the range of 100-300 pm. Moreover, a ratio of the (maximum) subdivider width (d6) at a given longitudinal position (in the bubble trap section) relative to the channel width (Wc) at the longitudinal position may, in embodiments, be selected from the range of 0.5-30, like from the range of 2-15. In further embodiments, the divider wall arrangement has a tapered shape at one or more of the extremes of the divider wall arrangement (i.e., at an upstream end and / or a downstream end of the divider wall arrangement). In embodiments, this may imply that the subdivider element at the upstream end and / or the subdivider element at the downstream end of the divider wall arrangement comprises that tapered shape (tapering to the fluid inlet and / or the fluid outlet, respectively. In further embodiments, the divider wall element may not comprise a fragmented divider wall, especially may not be fragmented, and one or more of the extremes of the (unfragmented) divider wall arrangement has the tapered shape.

[0106] If the divider wall arrangement comprises a non-fragmented divider wall (arrangement) (comprising no gaps), this may also be indicated with k=l. Moreover, in embodiments, a (maximal) width of the divider wall arrangement may be selected from values described herein for the width of the subdivider element. Furthermore, a length of the divider wall arrangement comprising an unfragmented divider wall may, in embodiments, be in the same range as a total length of the fragmented divider wall. In embodiments, e.g. a (total) length of the divider wall arrangement may be selected from the range of 300 pm - 20000 pm, like 500 pm - 15000 pm, especially 1000 pm - 10000 pm, and in embodiments 5000 pm -15000 pm. A height of the subdivider element may, in embodiments, be equal to the divider wall arrangement height (HSD). A (total) length of (the microfluidic channel) in the bubble trap section may, in embodiments, correspond to the length of the divider wall arrangement.

[0107] As described above, the divider wall arrangement is, in embodiments, configured parallel to the bubble trap section axis (EBT). The bubble trap section axis EBT) may, in embodiments, be configured substantially linear, especially the bubble trap section axis (EBT) may define a straight line. The bubble trap section axis (EBT) of elongation may be a straight axis of elongation, in embodiments. Additionally, or alternatively, the bubble trap section axis (EBT) may, in embodiments, comprise a curved or bent axis. Furthermore, the bubble trap wall section may, in embodiments, comprise protrusions extending in the direction of the divider wall arrangement. The (extremes of) the protrusions may, in embodiments, in combination with the divider wall arrangement define one or more of the (primary, secondary, or intermediate) first passage flow-through areas and / or one or more of the (primary, secondary, or intermediate) second passage flow-through areas.

[0108] In further specific embodiments, the divider wall arrangement is configured parallel to the bubble trap section axis (EBT) of elongation, wherein the bubble trap wall section comprises protrusions extending in the direction of the divider wall arrangement, wherein (extremes of) the protrusions in combination with the divider wall arrangement define (i) the primary first passage flow-through area, (ii) the secondary first passage flow-through area, (iii) the primary second passage flow-through area, and (iv) the secondary second passage flow- through area.

[0109] In further embodiments, partitions of the bubble trap wall section between the protrusions optionally define the intermediate first passage flow-through area and the intermediate second passage flow-through area.

[0110] As is described above (especially based on the described configurations), specific embodiments of (the microfluidic channel in) the bubble trap section may especially be configured asymmetrically. Such asymmetric configuration may facilitate trapping of bubbles.

[0111] The microfluidic device of the invention may in an aspect be used for a microfluidic application with a liquid (without gas bubbles). In specific embodiments, the microfluidic device may be used for membrane formation with a liquid (without gas bubbles). The membrane formation section is, in embodiments, especially configured downstream from the bubble trap section. Furthermore, the membrane formation section is, in further embodiments, especially configured upstream of the first fluid outlet. Hence, in embodiments, upstream of the membrane formation section, bubbles in the liquid may be trapped in the bubble trap section, as discussed above. In further embodiments, a number of bubbles in a liquid provided to the microfluidic device may be reduced, especially based on the bubble trap (section).

[0112] Such microfluidic device may, in embodiments, comprise more than one microfluidic channel. In specific embodiments, especially (only) one of these microfluidic channels may comprise the membrane formation section. For reasons of explaining the microfluidic device, herein it may be described that the first microfluidic channel may comprise the membrane formation section (and especially a further microfluidic channel may not comprise the membrane formation section). It will be understood that, in (further) embodiments, the second microfluidic channel may comprise the membrane formation section (instead of the first microfluidic channel or next to the first microfluidic channel).

[0113] Moreover, in embodiments, the second microfluidic channel may comprise a (second) bubble trap section configured downstream of the second fluid inlet and upstream of the one or more connection channels.

[0114] Furthermore, in embodiments, the width of the microfluidic channel may vary along the microfluidic channel axis. The width may for instance be a function of the longitudinal position in the bubble trap section as described above. Furthermore, in embodiments, the (average) channel width in the bubble trap section may further differ from the (average) width of the channel in the membrane formation section. Especially, in embodiments, the average channel width in the bubble trap section may be configured larger than in locations of the channel downstream of the bubble trap section. This may, in embodiments, result in a reduced flow rate in the bubble trap section relative to the membrane formation section, which may help trapping of bubbles, in embodiments. Conversely, in further embodiments, the channel width (Wc) at locations upstream of the bubble trap may be larger than the average channel width (Wc) in the bubble trap section. The channel width (Wc) at locations upstream of the bubble trap may, in embodiments, be constant and e.g. be the same as the channel width (Wc) at the upstream end of the bubble trap section.

[0115] In further specific embodiments, a maximal channel width (Wc) in the membrane formation section may be smaller than a maximal channel width (Wc) in the bubble trap section.

[0116] In further embodiments, the microfluidic device may further comprise a first exit, a second exit, and further connection channels. Moreover, in embodiments, the first exit and the second exit may each be configured fluidically connected to the first microfluidic channel via the(ir respective) further connection channels. Hence, in embodiments, the further connection channels may be defined as described above in relation to the first and second connection channels. Especially, in embodiments, the further connection channels may comprise similar connection channels as the first and second connection channels, but configured to connect the first microfluidic channel to the first and / or second exits (rather than connecting the first microfluidic channel to the second microfluidic channel). Hence, in embodiments, the further connection channels may be configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from one microfluidic channel (especially the first microfluidic channel) to the first and / or second exit. The further connection channels, may provide symmetry in the flow which may be beneficial for the formation of planar lipid bilayers. The first exit and second exit may provide further controllability of the fluid flow through the microfluidic device. By sealing different combinations of the first fluid outlet, the second fluid outlet, the first exit, and the second exit, the flow path of fluids through the microfluidic device may be adjusted. Such embodiments may be beneficial as they may provide versatility to the microfluidic device allowing it to be applied in a variety of different experiments. Furthermore, the first exit and the second exit may help balance the flow of fluids during membrane formation and improve flow control during replacement of the media at the formed membranes.

[0117] In a further aspect, the invention may provide a method for the formation of artificial cell lipid bilayers. In embodiments, the method may comprise a first stage comprising providing organic solvent comprising lipids to the first fluid inlet of the microfluidic device. The first stage may further, in embodiments, comprise subsequently providing a flow of (first) aqueous phase (optionally comprising microbeads) to the first fluid inlet of the microfluidic device to obtain an aqueous phase-organic phase interface (between the organic solvent and the (first) aqueous phase). Furthermore, in embodiments, the method may comprise a second stage comprising applying a pressure to the first fluid inlet (at least) until the aqueous phase-organic phase interface may be forced past the membrane formation section via the n+1 elongated subchannels. Hence, in specific embodiments, the invention may provide a method for the formation of artificial cell lipid bilayers, wherein the method may comprise: (i) a first stage comprising providing organic solvent comprising lipids to the first fluid inlet of the microfluidic device as described herein; and subsequently providing a flow of (first) aqueous phase (optionally comprising microbeads) to the first fluid inlet of the microfluidic device to obtain an aqueous phase-organic phase interface (between the organic solvent and the (first) aqueous phase); and (ii) a second stage comprising applying a pressure to the first fluid inlet (at least) until the aqueous phase-organic phase interface may be forced past the membrane formation section via the n+1 elongated subchannels. Such a method may provide the benefit of enabling users to conveniently form artificial cell membranes of various compositions in a high-throughput and relatively simple manner. No special skills may be required for the application of the method. Using only two conventional syringe pumps and the relatively fast and straightforward method steps described herein, artificial cell membranes may be formed.

[0118] In embodiments, the method may thus comprise providing an organic solvent comprising lipids to the first fluid inlet of the microfluidic device. Hence, in embodiments, the organic solvent comprising lipids may be provided to the first microfluidic channel, i.e., to the channel comprising the membrane formation section. In embodiments, the organic solvent may be any organic solvent, especially any organic solvent in which the desired lipids for the lipid bilayer may be dissolved. For example, in embodiments, the organic solvent may comprise one or more of chloroform, methanol, ethanol, butanol, isopropanol, chloroform, n-hexane, decane, acetone, benzene, cyclohexane, cyclopentyl methyl ether, 2-methyltetrahydrofuran and isopentyl acetate. In specific embodiments, the organic solvent may comprise at least chloroform. The choice for the organic solvent may, in embodiments, depend on the desired solvability for the lipids that may be used. Suitable combinations of organic solvents and lipids may be clear to the skilled person.

[0119] Especially, in embodiments, a volume X of organic solvent comprising lipids may be provided to the first inlet of the microfluidic device. The volume X may especially be related to (or depend on) the microfluidic channel dimensions, i.e., a larger channel may require (or facilitate or host) a larger volume of organic solvent, whereas a smaller channel may (facilitate or host or) be limited to a smaller volume of organic solvent. Furthermore, the volume X may be related to a flow velocity of the organic solvent during operation of the microfluidic device. In embodiments, the volume X may be selected from the range of >0.1 pL, such as from the range of >0.3 pL, like from the range of >0.5 pL. Especially, in embodiments, the volume X may be selected from the range of >0.6 pL, such as from the range of >0.8 pL, like from the range of >1 pL. Furthermore, in embodiments, the volume X may be selected from the range of <100 pL. Especially, in embodiments, the volume X may be selected from the range of <50 pL, such as from the range of <30 pL, like from the range of <15 pL, especially from the range of <5 pL. In some embodiments, the volume X may for example be selected from the range of 0.3-0.6 pL. In other embodiments, e.g. with a larger n*m array of the membrane formation section, the volume X may for example be selected from the range of 1-25 pL. Hence, in specific embodiments, providing organic solvent comprising lipids to the first inlet of the microfluidic device may comprise providing a volume X selected from the range of 0.3-100 pL of organic solvent comprising lipids. Such embodiments may be beneficial as the volume may be enough to form membranes with relatively high efficiency, while preventing damage of the microfluidic device by an overdose of the organic solvent.

[0120] As described, in embodiments, the organic solvent may comprise the lipids that may be used for lipid bilayer formation. In embodiments, the lipids may for example be selected from the group comprising: phospholipids, glycolipids, and cholesterol. Especially, in embodiments, the organic solvent may comprise a combination of different lipids, such as a combination of phospholipids, glycolipids, and cholesterol. More especially, in embodiments, the lipids may comprise one or more of l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), Palmitoyl-myristoyl-PC (PMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1- myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), l-palmitoyl-2-linoleoyl-sn- glycero-3 -phosphocholine (PLPC), l-palmitoyl-2-oleoyl-glycero-3 -phosphocholine (POPC), 1 -stearoyl -2-oleoyl-sn-glycero-3 -phosphocholine (SOPC), l,2-Diphytanoyl-3-sn- phosphatidylcholine (DPhPC), 3P-Hydroxy-5-cholestene, 5-Cholesten-3P-ol (cholesterol). Note, however, that essentially any lipid (especially ones that may form a lipid bilayer) may be used. Hence, the invention may not be limited to the herein exemplified lipids.

[0121] The method may further, in embodiments, comprise providing a flow of first aqueous phase to the first fluid inlet of the microfluidic device. The first aqueous phase may especially be added subsequently to the organic solvent to obtain an aqueous phase-organic phase interface (between the organic solvent and the (first) aqueous phase). In doing so, a lipid monolayer may form at the aqueous phase-organic phase interface.

[0122] In embodiments, the first aqueous phase may comprise one or more components selected from the group comprising: solvents, buffers, (inorganic) bases, salts, and (stabilizing) agents. For example, in embodiments, the first aqueous phase may comprise one or more buffers such as 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 2-(N- morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and phosphate-buffered saline (PBS)), 2-Amino-2-hydroxymethyl-propane-l,3-diol (TRIS), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES). Further, in embodiments, the first aqueous phase may comprise one or more (inorganic) bases such as e.g. concentrated KOH. Yet further, in embodiments, the first aqueous phase may comprise one or more salts such as e.g. KC1. Yet further, in embodiments, the first aqueous phase may comprise one or more stabilizing agents such as e.g. bovine serum albumin (BSA), sugars, sugar alcohols, and amino acids. In specific embodiments, the first aqueous phase may further comprise microbeads, such as e.g. polystyrene or silica microbeads.

[0123] Especially, in embodiments, at least 10 pL, such as at least 12 pL, especially at least 15 pL of first aqueous phase may be provided to the first inlet of the microfluidic device. Furthermore, in embodiments, at most 60 pL, such as at most 50 pL, like at most 40 pL, especially at most 30pL of first aqueous phase may be provided to the first inlet of the microfluidic device. For example, in embodiments, between 12-19 pL of first aqueous phase may be provided to the first inlet of the microfluidic device. Alternatively, in embodiments, for example between 25-50 pL of first aqueous phase may be provided to the first inlet of the microfluidic device. The volume of first aqueous phase may especially be related to (or depend on) the microfluidic channel dimensions, i.e., a larger channel may require (or facilitate or host) a larger volume of first aqueous phase, whereas a smaller channel may (facilitate or host or) be limited to a smaller volume of first aqueous phase. Furthermore, the volume of first aqueous phase may be related to a flow velocity of the first aqueous phase during operation of the microfluidic device. Yet further, the volume of first aqueous phase may be related to the number m-1 of fluidic connection sections (i.e. the number of membranes to be formed) and the membrane formation distance (dm).

[0124] Especially, in embodiments, enough first aqueous phase may be provided to the first microfluidic channel, such that the flow of fluids may surpass the micropillars configured closest to the first fluid outlet. More especially, in embodiments, enough first aqueous phase may be provided to the first microfluidic channel, such that the aqueous phase-organic phase interface may have passed (most, but preferably) all of the micropillars configured in the first microfluidic channel.

[0125] The method may further, in embodiments, comprise the second stage comprising applying a pressure to the first fluid inlet (at least) until the aqueous phase-organic phase interface may be forced past the membrane formation section. Especially, the aqueous phase- organic phase interface may be forced past the membrane formation section via the n+1 elongated subchannels (of the membrane formation section). In embodiments, such a pressure as described here may for example be provided by providing more of the first aqueous phase to the first microfluidic channel. Additionally or alternatively, in embodiments, such a pressure as described here may for example be provided by providing a second aqueous phase, see also further below. Yet additionally or alternatively, such a pressure as described here may for example be provided by providing another fluid, such as e.g. a gas or water, to the first microfluidic channel. In embodiments, when the lipid monolayer of the aqueous phase-organic phase interface may be pushed (e.g. by pressure of more of the first aqueous solvent being provided to the first microfluidic channel) to the micropillars, the micropillars may separate the flow of fluids into separate subflows for each of the n+1 elongated subchannels. Subsequently, in embodiments, every time the subflows reach a fluidic connection section, adjacent subflows may again be fluidically connecting, upon which the lipid monolayers of the fluidically connecting subflows may form a lipid bilayer. These steps of separating the flow into subflows and reconnecting the subflows to form a lipid bilayer may, in embodiments, be repeated at every micropillar that the flow of fluids may pass as a result of ongoing pressure (e.g. of more of the first aqueous phase being provided to the first microfluidic channel).

[0126] The method may further comprise trapping bubbles in the bubble trap section to provide a bubble free flow of the liquids to the membrane formation section.

[0127] Especially, in yet another aspect, the invention may further provide a method for reducing a number of bubbles in a liquid (“bubble reduction method”). The bubble reduction method may, in embodiments, comprise providing a microfluidic device described herein; and introducing a liquid via the (first and / or second) fluid inlet in the (first and / or second) microfluidic channel and flowing the liquid (especially comprising one or more bubbles) through one or more of the microfluidic channels (fluidically connected to the (first and / or second) fluid inlet). The term “bubbles” especially refers to gas bubbles.

[0128] The term “a liquid” may refer to a plurality of (different) liquids. For instance, in embodiment two or more different liquids may be introduced sequentially in the microfluidic channel. Moreover, in further embodiments, the microfluidic device may comprise two microfluidic channels and the same liquid is provided in both microfluidic channels. It will be understood that many different combinations are feasible.

[0129] In embodiments, the microfluidic device comprises the first microfluidic channel, and especially the liquid (such as a first liquid) is introduced in the first microfluidic channel via the first fluid inlet. In further embodiments, the microfluidic device comprises the second microfluidic channel, and especially the liquid (or a further liquid) may be introduced in the second microfluidic channel via the second fluid inlet.

[0130] In embodiments, the microfluidic device, especially the fluid outlet of the microfluidic device may be fluidly connected to a further microfluidic device and a (substantially) bubble free liquid may be provided to (an inlet of) the further microfluidic device. Especially, in embodiments, the microfluidic device may comprise the membrane formation section, and the (substantially) bubble free liquid is provided to the membrane formation section.

[0131] In a yet further aspect, the invention may provide a method for (high-throughput) active compound screening on artificial lipid bilayers. In embodiments, the method may comprise a first stage comprising providing lipid bilayers in the microfluidic device, for example through the above described method. In further embodiments, the method may comprise a second stage comprising after lipid bilayer formation providing a flow of second aqueous solvent to the microfluidic device to provide the active compound to the formed lipid bilayers. Yet further, in embodiments, the method may comprise a third stage comprising analyzing the lipid bilayer and / or the active compound (using optical characterization techniques) during the second stage. Additionally or alternatively, in embodiments, the third stage may comprise analyzing the lipid bilayer and / or the active compound (using optical characterization techniques) after the second stage. Hence, in specific embodiments, the invention may provide a method for (high-throughput) active compound screening on artificial lipid bilayers, wherein the method may comprise (i) a first stage comprising providing lipid bilayers in the microfluidic device (by performing the above described method); (ii) a second stage comprising after lipid bilayer formation providing a flow of second aqueous phase to the microfluidic device to provide the active compound to the formed lipid bilayers (via the one or more connection channels); and (iii) a third stage of comprising analyzing the lipid bilayer and / or the active compound (using optical characterization techniques) during the second stage and / or after the second stage.

[0132] Such embodiments may be beneficial as the method may enable users to conveniently form artificial cell membranes of various compositions in a high-throughput manner and perform drug screening tests on them. Hence, with such a method it may be possible to test drugs on artificial cell membranes in-vitro in a relatively simple manner. No special skills may be required for application of the method. Using e.g. conventional syringe pumps and following a fast and straightforward protocol artificial cell membranes may be formed and may be tested. In this way, the effect of drugs (e.g. antibiotics), proteins, toxins, microplastics, and any other type of reagents may be screened on cell membranes in-vitro in a robust and high-throughput manner.

[0133] All the steps of forming membranes, treating them with drug, performing measurements and characterizations, and doing (fluorescent) imaging may be done all on one (kit comprising the) microfluidic device using the method described herein. The method may require only the use of a single microfluidic device to form and test relatively high numbers of membranes (e.g. up to 30 membranes) simultaneously (within less than 2 minutes), therewith decreasing costs and required time for high-throughput experiments.

[0134] Furthermore, the method may provide the benefit of flexibility, as the microfluidic device applied in the method may be compatible with various different tests and experiments that may need to be performed on membranes, such as e.g., fluorescent and confocal microscopy, optical tweezers, etc. Yet further, due to the material used to fabricate the microfluidic device, even harsh chemicals may be used as solvents in the method.

[0135] Especially, in embodiments, the first stage may comprise, after the above described step of providing organic solvent comprising lipids to the first fluid inlet, providing a flow of first aqueous phase (optionally comprising microbeads) to the first fluid inlet. As a result, in embodiments, lipid bilayers may be obtained between the micropillars. Subsequently, in embodiments, the first stage may comprise providing a flow of second aqueous phase comprising an active compound to the second fluid inlet. The flow of second aqueous phase may, in embodiments, be halted when one of the one or more connection channels (especially the first connection channel) may be reached. In further embodiments, the method may comprise a second stage comprising after lipid bilayer formation blocking the second fluid outlet and halting the flow of first aqueous phase. Subsequently, in embodiments, the second stage may comprise restarting the flow of second aqueous phase to provide the active compound to the formed lipid bilayers (via the one or more connection channels).

[0136] Furthermore, in embodiments, the method may comprise (i) a first stage comprising providing lipid bilayers in the microfluidic device as described above; (ii) a second stage comprising after lipid bilayer formation (iia) halting the flow of first aqueous phase, and (iib) providing a flow of second aqueous phase comprising an active compound to the microfluidic device to provide the active compound to the formed lipid bilayers; and (iii) a third stage comprising analyzing the lipid bilayer and / or the active compound (using optical characterization techniques) during the second stage and / or after the second stage. Furthermore, in such embodiments, the first stage may comprise providing lipid bilayers in the microfluidic device by (ia) providing organic solvent comprising lipids to the first fluid inlet, and subsequently providing a flow of first aqueous phase (optionally comprising microbeads) to the first fluid inlet to obtain lipid bilayers between the micropillars; and (ib) providing a flow of second aqueous phase comprising the active compound to the second fluid inlet, and halting the flow of second aqueous phase when one of the one or more connection channels (especially the first connection channel) may be reached. In such embodiments, the second stage may further comprise after lipid bilayer formation comprising (iia) prior to halting the flow of first aqueous phase blocking the second fluid outlet, (iib) halting the flow of first aqueous phase, and, after halting the flow of first aqueous phase, (iic) restarting the flow of second aqueous phase to provide the active compound to the formed lipid bilayers (via the one or more connection channels). Hence, in specific embodiments, the method may comprise (i) the first stage comprising providing lipid bilayers in the microfluidic device (by performing the method described herein); wherein the first stage may comprise (ia) after providing organic solvent comprising lipids to the first fluid inlet, providing a flow of first aqueous solvent (optionally comprising microbeads) to the first fluid inlet to obtain lipid bilayers between the micropillars; and (ib) providing a flow of second aqueous solvent comprising an active compound to the second fluid inlet, and halting the flow of second aqueous solvent when one of the one or more connection channels (especially the first connection channel) may be reached; and (ii) the second stage comprising after lipid bilayer formation (iia) blocking the second fluid outlet, (iib) halting the flow of first aqueous solvent, and (iic) restarting the flow of second aqueous solvent.

[0137] Embodiments of the organic solvent and the first aqueous phase have been described above. Furthermore, in embodiments, the second aqueous phase may comprise one or more components selected from the group comprising: buffers, (inorganic) bases, salts, and (stabilizing) agents. For example, in embodiments, the second aqueous phase may comprise one or more buffers such as 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 2- (N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), phosphate-buffered saline (PBS)), 2-Amino-2-hydroxymethyl-propane-l,3-diol (TRIS), and Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES). Further, in embodiments, the second aqueous phase may comprise one or more (inorganic) bases such as e.g. concentrated KOH. Yet further, in embodiments, the second aqueous phase may comprise one or more salts such as e.g. KC1. Yet further, in embodiments, the second aqueous phase may comprise one or more stabilizing agents such as e.g. bovine serum albumin (BSA), sugars, sugar alcohols, and amino acids. In specific embodiments, the second aqueous phase may comprise a solvent compatible with (i.e. capable of dissolving without detrimentally affecting) the active compound.

[0138] Especially, in embodiments, at least 5 pL, such as at least 10 pL, especially at least 15 pL of second aqueous phase may be provided to the microfluidic device. Furthermore, in embodiments, at most 60 pL, such as at most 40 pL, like at most 20 pL of second aqueous phase may be provided to the microfluidic device. For example, in embodiments, between 4- 16 pL of second aqueous phase may be provided to the microfluidic device. Alternatively, in embodiments, for example between 25-50 pL of second aqueous phase may be provided to the microfluidic device. In embodiments, the second aqueous phase may thus comprise an active compound. Especially, the active compound may comprise any molecule of interest for molecule-membrane interaction experiments. For example, in embodiments, the active compound may comprise one or more of a drug, a protein, an amino acid, a biomarker, a fluorophore (or fluorescent dye), an antibiotic, a hormone, a lipid, a carbohydrate such as a sugar, a salt, a nucleic acid, and any other membrane interacting agent. Especially, in embodiments, the active compound may comprise one or more of a drug, a protein, an antibiotic, and a fluorophore. In specific embodiments, the active compound may comprise a drug and / or an antibiotic.

[0139] The different fluids, such as the organic solvent, the first aqueous phase, and the second aqueous phase may, in embodiments, be provided to any one of the inlets. Especially, in embodiments, the organic solvent may be provided to the first fluid inlet. Similarly, in embodiments, the first aqueous phase may be provided to the first fluid inlet. In some embodiments, the second aqueous phase may also be provided to the first fluid inlet, e.g. when a microfluidic device is used having only the first microfluidic channel (and thus not the second microfluidic channel). However, in alternative embodiments, the second aqueous phase may be provided to the second fluid inlet. Providing the fluids to the fluid inlets may, e.g., be achieved through the use of (syringe) pumps. In embodiments, (syringe) pumps may be configured to provide and manipulate (e.g. halt or restart) the flow of the different fluids into the microfluidic device. Furthermore, in embodiments, (syringe) pumps may be configured to control the flow (e.g. flow velocity and volume) of fluids into the microfluidic device.

[0140] In embodiments, the flow of second aqueous phase may be halted when one of the one or more connection channels (especially the first connection channel) may be reached. Such embodiments may be beneficial as starting the flow of the second aqueous phase simultaneously with that of the first aqueous phase may reduce disturbance of the lipid bilayers (when configuring a connection from a pump to the fluid inlet) as compared to starting the flow of the second aqueous phase after lipid membrane formation. Furthermore, by halting the flow of the second aqueous phase when one of the one or more connection channels (especially the first connection channel) may be reached, one may allow lipid bilayer formation without corrupting the formation process with the active compound in the second aqueous phase.

[0141] To assist with flow manipulation, in embodiments, one or more of the first fluid outlet, the second fluid outlet, the first exit, and the second exit may be blocked. In embodiments, blocking of such outlets or exits may be achieved though e.g. plugging the opening using a glue (e.g. adhesive resin), grease (e.g. silicon), or by taping shut the opening. Hence, in exemplary embodiments, the working principle of (i.e. applying the method to) the microfluidic device may comprise flowing chloroform containing lipid molecules into the device from the first fluid inlet, followed by flowing an aqueous media that may also be referred to as “aqueous phase I” into that same first fluid inlet. The aqueous phase I may, in some embodiments, consists of e.g. HEPES, optionally with other components such as microbeads (e.g. if the device is to be used in an optical tweezers experiment). Further, in embodiments, from the second fluid inlet, the second aqueous phase (or replacement media or “aqueous phase II”) may be flown into the second microfluidic channel. The flow in the first microfluidic channel may, in embodiments, continue to be pushed forward in the direction of elongation, while the flow of second aqueous phase (or replacement flow) may be stopped by the time its flow front reaches the first connection channel. In embodiments, any unwanted bubble in either channel may be trapped in the bubble traps configured in the first microfluidic channel and the second microfluidic channel and may thus be removed from the flow of fluids heading further downstream of the channels, therewith providing the device with bubble-free flows. Then, by pushing further the flow in the first microfluidic channel, in embodiments, the flow may reach the membrane formation section comprising the micropillars. In such embodiments, the flow may be partitioned into n+1 subflows when reaching the n rows of micropillars. In embodiments, when the n+1 subflows reach a fluidic connection section, then the subflows may join again. In the rejoining of the subflows, two lipid monolayers may be joined and may zip with one another to form lipid bilayers, i.e. artificial cell membranes. In embodiments, the partitioning-zipping may continually occur at every micropillar that the flow may pass, such that at every fluidic connection section a lipid bilayer may be formed. At this stage, in embodiments, a part of the first aqueous phase may, due to increasing pressure, flow through one of the further connection channels toward the first exit. Additionally or alternatively, in such embodiments, a part of the first aqueous phase may, due to increasing pressure, flow through one of the first connection channel and second connection channel to the second microfluidic channel. In embodiments, the amount of first aqueous solution flowing into the connection channels may be relatively low, due to a substantial pressure drop that may be induced by the narrow cross-sectional circular diameter (DRC) and the total (e.g. zig-zag) channel length (LRC) of the connection channel.

[0142] In embodiments, once the lipid bilayers are formed, the flow of first aqueous solution into the first microfluidic channel may be stopped and the flow of the second aqueous solution (aqueous phase II, which contains e.g. drugs) may be (re-)started. The second aqueous solution may initially wash away all the fluid that flowed from the first microfluidic channel to the second microfluidic channel (via the connection channels) out to the second fluid outlet, after which the second fluid outlet may be blocked (e.g. sealed using a sealing agent / resin). Subsequently, in embodiments, the second aqueous solution may be pushed through the connection channels and may enter the first microfluidic channel, especially the membrane formation section. As such, in embodiments, the fluid around the lipid bilayers may be replaced and the contents of second aqueous solution (e.g. the drug or active component) may be delivered to the lipid bilayers.

[0143] Afterwards, in embodiments, the first microfluidic channel may be washed free of second aqueous solution and refilled with first aqueous solution by (i) stopping the flow of second aqueous solution into the second microfluidic channel, (ii) restarting the flow of first aqueous solution into the first microfluidic channel, and (iii) sealing both the first and second exits. Such embodiments may be useful for example for delivering fluorescent molecules to membranes and then washing those molecules from the channel after a predetermined time.

[0144] In embodiments, the third stage may comprise analyzing the lipid bilayers that have formed in the microfluidic device. Additionally or alternatively, the third stage may comprise analyzing the interaction of the active compound with the lipid bilayers that have formed in the microfluidic device. Therefore, in embodiments, the third stage may comprise applying analysis techniques, especially optical characterization techniques. Especially, in embodiments, the optical characterization techniques may comprise one or more of the group comprising: optical tweezer trapping, particle tracking, laser-induced fluorescent testing, confocal microscopy, total internal reflection fluorescence microscopy, fluctuation analysis, and Raman spectroscopy.

[0145] The third stage comprising analyzing the lipid bilayer and / or the active compound may especially be performed during the second stage. Additionally, or alternatively, the third stage comprising analyzing the lipid bilayer and / or the active compound may also be performed after the second stage.

[0146] In a yet further aspect, the invention may provide a kit of parts for the formation of lipid bilayers. Especially, in embodiments, the kit of parts may comprise the microfluidic device, a holder comprising an organic solvent (such as e.g. the ones described above), and a holder comprising a first aqueous phase (such as e.g. the ones described above). Further, in embodiments, the kit of parts may comprise one or more syringe pumps for providing the organic solvent and the first aqueous phase to the microfluidic device. Yet further, in embodiments, the kit of parts may comprise a holder comprising a second aqueous phase (such as e.g. the ones described above). Furthermore, in embodiments, the holder comprising the organic solvent may optionally also comprise lipids. However, in alternative embodiments, the kit of parts may comprise a separate holder comprising the lipids. Similarly, in embodiments, the holder comprising the first aqueous phase may optionally also comprise microbeads. However, in alternative embodiments, the kit of parts may comprise a separate holder comprising the microbeads. Hence, in specific embodiments, the invention may provide a kit of parts for the formation of lipid bilayers, comprising: the microfluidic device, a holder comprising an organic solvent; and a holder comprising a first aqueous phase.

[0147] Yet in a further aspect, the invention may provide a system for performing optical measurements on artificial lipid bilayers. Especially, the system may comprise the microfluidic device. Moreover, in embodiments, the system may comprise a fluid managing device (such as e.g. a pump) configured fluidically connected to the microfluidic device. In embodiments, the fluid managing device may be configured to provide fluids (such as one or more of the organic solvent, the first aqueous phase, and the second aqueous phase) to the first fluid inlet and / or second fluid inlet, of the microfluidic device. Additionally or alternatively, in embodiments, the fluid managing device may be configured to dispose of fluids exiting from one or more of the first fluid outlet, the second fluid outlet, the first exit, and the second exit. In further embodiments, the system may comprise an optical measurement device. In embodiments, the optical measurement device may be configured to provide radiation to the microfluidic device. Additionally or alternatively, in embodiments, the optical measurement device (such as e.g. an optical microscope or a Lumicks C-Trap®) may be configured to detect radiation emitted from the microfluidic device. Hence, in specific embodiments, the invention may provide a system for performing optical measurements on artificial lipid bilayers, wherein the system may comprise: (A) the microfluidic device as described herein; (B) a fluid managing device configured to (i) provide fluids to the first fluid inlet and / or second fluid inlet of the microfluidic device, and (ii) dispose of fluids exiting from one or more of the first fluid outlet, the second fluid outlet, the first exit, and the second exit; and (C) an optical measurement device configured to (i) provide radiation to the microfluidic device, and / or (ii) detect radiation emitted from the microfluidic device.

[0148] BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Figs. 1 schematically depict an embodiment of the microfluidic device of the invention. Figs. 2-6 schematically depict some aspects of the invention. Fig. 7 schematically depicts an embodiment of the system of the invention. The schematic drawings are not necessarily on scale.

[0150] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0151] Fig. 1 schematically depicts a microfluidic device 100 for the formation of lipid bilayers. In embodiments, the microfluidic device 100 may comprise a first fluid inlet 111, a first fluid outlet 112, and a first microfluidic channel 110 configured to fluidically connect the first fluid inlet 111 and the first fluid outlet 112. In further embodiments, the microfluidic device may comprise two microfluidic channels 109, see e.g. Fig. 4. Herein, these two microfluidic channels 109 may (also) be indicated as the first microfluidic channel 110 and the second microfluidic channel 120. Moreover, in embodiments, comprising (only) a first microfluidic channel 110, the first microfluidic channel 110 may also be indicated as microfluidic channel 109. Moreover, the terms “first fluid inlet” 111 and “first fluid outlet” 112, may also be used referring the fluid inlet 101 and the fluid outlet 102 respectively of the microfluidic channel 109. Likewise, the second fluid inlet 121 and the second fluid outlet 122 of the second microfluidic channel 120 may also be referred to as fluid inlet 101 and fluid outlet 102 of the microfluidic channel 109 (see e.g. Fig. 4).

[0152] Further, in embodiments, the first microfluidic channel 110 may comprise a channel wall 115 defining a channel height (He, see Fig. 1 subfigure II). Yet further, in embodiments, the first microfluidic channel 110 may comprise a membrane formation section 200 having a membrane formation section axis EmfSof elongation. In embodiments, the channel wall 115 at the membrane formation section 200 may comprise a membrane formation wall section 215. The membrane formation section 200 may, in embodiments (such as highlighted in the cut-out in Fig. 1 and in Fig. 2), comprise a plurality of micropillars 210. Especially, each of the plurality of micropillars 210 may be configured to extend along the (full) channel height He. Especially, the plurality of micropillars 210 may be configured in an n*m array parallel to the membrane formation section axis EmfSof elongation. Especially, n may be selected from the range of >1 (n=2 as depicted in Fig. 1) and m may be selected from the range of >2 (m=15 as depicted in Fig. 1). Further, in embodiments, the n*m array may be configured to define n+1 (parallelly configured) elongated subchannels 110a, 110b, 110c... within the first microfluidic channel 110. Additionally, in embodiments, the n*m array may be configured to define per row of the n rows m-1 fluidic connection sections 105. Further, in embodiments, adjacent elongated subchannels 110a, 110b, 110c... may be configured fluidically connected at the m-1 fluidic connection sections 105 configured between adjacent micropillars 210.

[0153] As depicted in the cut-out in Fig. 1 and in Fig. 2, in embodiments, the plurality of micropillars 210 each may have a maximum width Wmax defined in a direction perpendicular to the membrane formation section axis Emfs of elongation (and perpendicular to the channel height He). Especially, in embodiments, the maximum width Wmax may be selected from the range of 50-300 pm. Moreover, in embodiments, the micropillars 210 may have a micropillar length Lmdefined parallel to the membrane formation section axis Emfs of elongation. Especially, in embodiments, the micropillar length Lmmay be selected from the range of 50- 1000 pm. Further, in embodiments, each of the plurality of micropillars 210 may taper from its maximum width Wmax at least in directions parallel to the membrane formation section axis Emfs of elongation towards respective adjacent micropillars 210. Especially, as depicted here, one or more of the plurality of micropillars 210 may have a diamond-like cross-sectional shape (in a plane perpendicular to the height He).

[0154] Fig. 1 subfigure II schematically depicts a side view of the microfluidic device 100 in a cross-sectional plane perpendicular to the membrane formation section axis Emfs of elongation as depicted in Fig. 1 subfigure I. As depicted here, in embodiments, the channel wall 115 may be faceted. Especially, in embodiments, the channel wall may comprise four facets. More especially, in such embodiments, the channel wall 115 may comprise a channel bottom section 140, a channel top section 150, and a spacer wall 160 configured to connect the channel bottom section 140 to the channel top section 150. Hence, in such embodiments, the spacer wall 160 may define the channel height He, especially, the channel height He may be defined between the channel bottom section 140 and the channel top section 150. Furthermore, in such embodiments, (where the microfluidic channel may have a rectangular (or polygonal) cross- sectional shape) the membrane formation wall section 215 may refer to the spacer wall 160 defined along the subchannel length Lsc.

[0155] Furthermore, as depicted in Fig. 2, a first distance dl may be defined between the membrane formation wall section 215 at the maximum width Wmax of the micropillars 210 and the membrane formation section axis Emfs of elongation. Conversely, a second distance d2 may be defined between the membrane formation wall section 215 at the fluidic connection sections 105 between the micropillars 210 and the membrane formation section axis Emfs of elongation. Furthermore, a third distance d3 may be defined between the membrane formation wall section 215 at the maximum width Wmax of the micropillars 210 and the micropillars 210 of an adjacent row m of the n*m array. Conversely, a fourth distance d4 may be defined between the membrane formation wall section 215 and the fluidic connection sections 105 between the micropillars 210 of that row m.

[0156] As depicted in Fig. 2 subfigure II, in embodiments, the membrane formation wall section 215 may be configured substantially linear. Hence, in such embodiments, the first distance dl may essentially be equal to both the second distance d2 and the fourth distance d4.

[0157] Alternatively, in embodiments, such as depicted in Fig. 2 subfigure I, the membrane formation wall section 215 may be configured in a (sinusoidal or triangular) wavelike shape parallel to the membrane formation section axis Emfs of elongation (see subfigure I). Especially, in embodiments as depicted in Fig. 1 and Fig. 2 subfigure I, the membrane formation wall section 215 may be configured in a (sinusoidal or triangular) wave-like shape mirroring the tapering of the plurality of micropillars 210 (in a row m adjacent to the wall 215). Hence, in such embodiments, the first distances dl may be smaller than the second distances d2. Similarly, in such embodiments, the third distances d3 may be smaller than the fourth distances d4.

[0158] Furthermore, in embodiments, the n+1 elongated subchannels 110a, 110b, 110c... may have a subchannel length Lsc defined as the largest distance between the micropillar 210 configured closest to the first fluid inlet 111 and the micropillar 210 of the same row m configured closest to the fluid outlet 112.

[0159] Moreover, in embodiments, the microfluidic device 100 may have a height H and a width W defined in a plane perpendicular to the membrane formation section axis Emfs of elongation (i.e. as shown in the figure the dimension extending perpendicularly from the drawing / paper), and a length L defined parallel to the membrane formation section axis Emfs of elongation. Additionally, or alternatively, in embodiments, the microfluidic device 100 may have a height H and a width W defined in a plane perpendicular to the bubble traps section axis EBT of elongation (i.e. as shown in the figure the dimension extending perpendicularly from the drawing / paper), and a length L defined parallel to the bubble trap section axis EBT of elongation. Especially, in embodiments, L>2*H and W>2*H.

[0160] In embodiments, the first microfluidic channel 110 may further comprise a (first) bubble trap 300 configured downstream of the first fluid inlet 111 and upstream of the membrane formation section 200.

[0161] Especially, as depicted in Fig. 1 and in Fig. 3, the microfluidic device 100 further may comprise a bubble trap. Moreover, the (first) microfluidic channel 110 comprises a bubble trap section 300 with a bubble trap section axis EBT of elongation (herein also indicated as “bubble trap section axis EBT“). Furthermore, the channel wall 115 at the bubble trap section 300 may comprises a bubble trap wall section as is indicated with reference number 315. The bubble trap wall section 315 may, in embodiments, extend along the channel height He. The depicted bubble trap section 300 further comprises a divider wall arrangement 310, dividing the (first) microfluidic channel 109(, 110) (in the bubble trap section 300) in a first passage 301 and a second passage 302. The passages 301,302 are configured parallel to the divider wall arrangement 310 and especially at opposite sides of the divider wall arrangement 310.

[0162] Herein also the term “bubble trap” may be used referring to the bubble trap section 300. The bubble trap 300 is especially configured for trapping bubbles 5 (see e.g. Fig. 6) in a flow provided to the (first) fluid inlet 101(, 111). The trapping of a bubble 5 may especially be the result of changing sizes of the flow-through area in the respective passages 301,302. The term flow-through area especially refers to an open cross-section in the microfluidic channel 109 through which the fluid may flow. For instance, if no divider wall arrangement 310 would be present in the bubble trap section 300 at a specific longitudinal position, then the flow-through area would be equal to the (inner) cross section of the microfluidic channel 109 at the specific longitudinal position. In figs. 1 and 3 multiple flow- through areas are indicated. These flow-through areas are indicated with the references An, A19, A12, A21, A29, and A22. An, A19, A12 refer to a primary first passage flow-through area, a largest intermediate first passage flow-through area, and a secondary first passage flow-through area, respectively; all configured in the first passage 301. A21, A19, and A22 refer to a primary second passage flow-through area, a largest intermediate second passage flow-through area, and a secondary second passage flow-through area, respectively; all configured in the second passage 302. The figure further depicts that the primary first passage flow-through area An is configured upstream of the secondary first passage flow-through area A12, and that the primary second passage flow-through area A21 is configured upstream of the secondary second passage flow-through area A22. Furthermore A 19 is configured upstream of A12 and downstream of An, and A29 is configured upstream of A22 and downstream of A21. In the depicted embodiment, An>A2i and An<A22. Moreover, in the depicted embodiment the primary ratio A21 / A11 is about 0.5 and the secondary ratio of Ai2 / A22 is about 0.5. Furthermore the intermediate passage flow through areas Ai9,A29 are both larger than the primary passage flow-through areas An,Ai2, and larger than the secondary passage flow-through areas A2i,A22 in the respective passages 301,302. Hence, A29 >A2i and A29 > A22, and A19 >An and A19 > A12 in the depicted embodiment. Furthermore, in the embodiment A19 / A29 is larger than 1. Furthermore, in embodiments, a first intermediate ratio A19 / A11 may be selected from the range of 1.5 to 5. Additionally, or alternatively a second intermediate ratio A29 / A21 may, in embodiments, be selected from the range of 1.5 to 5. Such dimensions may assist in trapping bubble in the bubble trap 300.

[0163] In Figs. 1 and 3, further two opposite wall portions 3151, 3152 of the bubble trap wall section 315 are indicated. The opposite wall portions 3151, 3152 may especially be arranged at opposite sides of the divider wall arrangement 310. In the depicted embodiment, a shortest distance d7 between two opposite wall portions 3151, 3152 of the bubble trap wall section 315 reduces in a direction from the fluid inlet 101 up to a first longitudinal position 316 (see also Fig. 3). The first longitudinal position 316 may, in embodiments, comprise the primary first passage flow-through area An. The first longitudinal position 316 may, in further embodiments, comprise the primary second passage flow-through area A21. In the depicted embodiment, the first longitudinal position 316 comprises the primary first passage flow- through area An and the primary second passage flow-through area A21, see also Fig. 3 in which the bubble trap wall section 300 of Fig. 1 is depicted further zoomed in. It is noted that, in alternative embodiments, the primary second passage flow-through area A21 may be configured closer to the fluid inlet 101 than the primary first passage flow-through area An.

[0164] In these figures, the shortest distance d7 between the two opposite wall portions 3151,3152 further increases (in the bubble trap section 300) in a direction from a second longitudinal position 317 to the fluid outlet 102. The second longitudinal position 317 in the depicted embodiment comprise the secondary first passage flow-through area A12 and the secondary second passage flow-through area A22.

[0165] Moreover, in such embodiments, the bubble trap wall section 315 may refer to the spacer wall 160 defined in the bubble trap section 300.

[0166] Fig. 1 and Fig. 3, further depict that the divider wall arrangement 310 may comprise a fragmented divider wall 310, in embodiments. The fragmented divider wall 310 in these figures comprises an array of four (aligned) subdivider elements 311 (configured parallel to the bubble trap section axis EBT. The four subdivider elements 311 are mutually separated by three gaps 312 (between the subdivider elements 311). The embodiment thus is an example of a fragmented divider wall comprising an array of k subdivider elements, wherein k = 4. In Fig. 1 subfigure II also a shortest distance d5 between adjacently configured subdivider elements 311 is indicated. Such shortest distance d5 may herein also be indicated as “gap size” d5. The gap size d5 is preferable configured to allow a liquid phase to flow between the two passages 301,302, and to prevent a bubble 5 to move between the passages 301,302. The gap size d5 may, in embodiments, be in the range of 10 to 150 pm, especially in the range of 50- 120 pm. Fig. 1 subfigure II and Fig. 3 further depict subdivider elements 311 having a subdivider length LSD, especially defined parallel to the bubble trap section axis EBT of elongation (or parallel to the divider wall arrangement 310). The subdivider length LSD may, in embodiments, be selected from the range of 100-3000 pm. The subdivider element 311 may, in embodiments, further comprise a (maximum) subdivider width d6 configured perpendicular to the subdivider length LSD (and to the channel height He (or divider wall arrangement height HSD). The width d6 may, in embodiments, refer to a maximum width d6, such as in the depicted embodiments, wherein the subdivider elements 311 taper towards their extremes. In the depicted embodiment, adjacently configured subdivider elements 311 have a tapering shape tapering in a direction towards each other. Furthermore in the embodiment (also) the divider wall arrangement 310 has a tapered shape at extremes of the divider wall arrangement 310 (i.e. at an upstream end and at a downstream end of the divider wall arrangement 310.

[0167] Furthermore, as depicted in Fig. 1 (and also in Fig. 4), in embodiments, a maximal width of the microfluidic channel 109,110 at the membrane formation section 200 (for instance configured at the upstream end of the membrane formation section 200) may be smaller than a maximal width of the microfluidic channel 109,110 at the bubble trap section 300 (for instance configured at the upstream end of the bubble trap section 300).

[0168] Especially, as depicted in Fig. 3, the divider wall arrangement 310 may be configured parallel to the bubble trap section axis EBT of elongation and especially the bubble trap section axis EBT of elongation is configured substantially linear (which may herein also be indicated as the axis of elongation comprises “a straight axis of elongation”). The bubble trap wall section 315 in Fig.3 comprises protrusions 320 extending in the direction of the divider wall arrangement 310. Moreover, (extremes of) the protrusions 320 in combination with the divider wall arrangement 310 define the primary first passage flow-through area An, the secondary first passage flow-through area An, the primary second passage flow-through area A21 and the secondary second passage flow-through area A22 in the depicted embodiment. Furthermore, Fig. 3 further depicts an embodiment wherein partitions of the bubble trap wall section 315 between the protrusions 320 define the intermediate first passage flow-through area A19 and the intermediate second passage flow-through area A29.

[0169] Moreover, as depicted in Fig. 4, in embodiments, the microfluidic device 100 may further comprise a second fluid inlet 121, a second fluid outlet 122, a second microfluidic channel 120 configured to fluidically connect the second fluid inlet 121 and the second fluid outlet 122, and one or more connection channels 400. In embodiments, the one or more connection channels 400 may be configured to fluidically connect the second microfluidic channel 120 to the first microfluidic channel 110.

[0170] In embodiments, the (first) microfluidic channel 109(, 110) may further comprise a (first) bubble trap 300 configured downstream of the (first) fluid inlet 101 (,111) and upstream of the membrane formation section 200, as is for instance depicted in Fig. 1. In that embodiment, the formation section axis Emfs of elongation and the bubble trap section axis EBT of elongation are aligned. In alternative embodiments, the formation section axis Emfs of elongation and the bubble trap section axis EBT of elongation may be configured at an angle to each other (yet both being configured in the same plane), as is for instance depicted in one of the microfluidic channels 109, i.e. the one indicated with reference 110, in Fig. 4.

[0171] Fig. 4 may at the same time also depict an embodiment comprising two microfluidic channels 109 (also indicated with references 110 and 120, referring to the first microfluidic channel and the second microfluidic channel, respectively), two fluid inlets 101 (also indicated with references 111 and 121 (for the first and second fluid inlet, respectively), and two fluid outlets 102 (indicated with references 112 and 122 for the first and second fluid outlet, respectively), wherein any one of the microfluidic channels 109 is configured to fluidically connect one of the fluid inlets 101 and a respective fluid outlet 102, wherein the microfluidic device 100 further comprises one or more connection channels 400 configured to fluidically connect the microfluidic channels 109 to each other, wherein the one or more connection channels 400 are configured downstream from the respective bubble trap sections 300.

[0172] Especially, the one or more connection channels 400 each have (i) a (connection) channel axis CA, (ii) a first joint 401 of the respective connection channel 400 and the first microfluidic channel 110 and (iii) a second joint 402 of the respective connection channel 400 and the second microfluidic channel 120. In embodiments, the connection channels 400 may be configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from one microfluidic channel (e.g. 120) to the other microfluidic channel (e.g. 110). Further, in embodiments, the one or more connection channels 400 each have a total channel length LRC defined along the (connection) channel axis CA between (i) the first joint 401 and (ii) the second joint 402. Especially, in embodiments, the total channel length LRC may be selected from the range of 2-50 mm. In further embodiments, the one or more connection channels 400 each have an equivalent cross-sectional circular diameter DRC defined perpendicular to the (connection) channel axis CA of the connection channel 400. In embodiments, the equivalent cross-sectional circular diameter DRC may be selected from the range of 50-500 pm.

[0173] In further embodiments, the one or more connection channels 400 may comprise a first connection channel 410 and a second connection channel 420. Especially, in embodiments, the first connection channel 410 may be configured upstream of the membrane formation section 200 (and downstream of the bubble trap). Moreover, in embodiments, the second connection channel 420 may be configured downstream of the membrane formation section 200.

[0174] The one or more connection channels 400 may, in embodiments, be configured essentially linear (not depicted). Alternatively, in embodiments, the one or more connection channels 400 may, in embodiments, be configured curved (not depicted). In specific embodiments, such as depicted in Fig. 4, at least one of the one or more connection channels 400 may be configured in a zigzag pattern.

[0175] In further embodiments, the second microfluidic channel 120 may comprise a (second) bubble trap section 300 configured downstream of the second fluid inlet 121 and upstream of the one or more connection channels 400.

[0176] In the depicted embodiment, each microfluidic channel 109 (110,120) comprises a channel wall 115 defining a respective channel height He. Furthermore, each microfluidic channel 109 (110,120) may comprise a respective bubble trap section 300 having a bubble trap section axis EBTI of elongation. It is noted that the respective bubble trap section axes EBTI of elongation may be configured at an angle. Moreover, the bubble trap section axes EBTI of elongation may be configured at an angle with the membrane formation section axis Emfs of elongation configured in the same microfluidic channel 109,110 as is depicted in the figure.

[0177] Further, for each of the microfluidic channels 109 (110,120) (i) the channel wall 115 at the bubble trap section 300 may comprise a respective bubble trap wall section 315 extending along the respective channel height He; (ii) the bubble trap section 300 may comprise a respective divider wall arrangement 310 configured to divide the respective microfluidic channel 109 (110, 120) in the bubble trap section 300 in a first passage 301 and a second passage 302; and (iii) the bubble trap wall section 315 and the respective divider wall arrangement 310 may define (a) a respective primary first passage flow-through area An and a respective secondary first passage flow-through area An (in the first passage 301), with the primary first passage flow-through area An being configured upstream of the secondary first passage flow- through area An, and (b) a respective primary second passage flow-through area A21 and a respective secondary second passage flow-through area A22 (in the second passage 301), with the primary second passage flow-through area A21 being configured upstream of the secondary second passage flow-through area A22. In embodiments, are the respective An, A12, A21, and A22 for any one of the microfluidic channels 109 independently from each other selected such that An>A2i and An<A22. Hence, in embodiment An (as well as A12, A21, A19, etc.) of the first microfluidic channel 110 and An(as well as A12, A21, A19, etc.) of the second microfluidic channel 120 may have a different value. Hence, each bubble trap section 300 in the respective microfluidic channels 109 may have been configured independently from each other as described in relation to the microfluidic device with a single microfluidic channel 109 (for instance as depicted in Fig. 1).

[0178] It is noted that Fig. 4 depicts an embodiment with two microfluidic channels 109. Yet further microfluidic device may have more than two microfluidic channels 109.

[0179] Yet further, in embodiments, the microfluidic device 100 may further comprise a first exit 10, a second exit 20, and further connection channels 405. In embodiments, the first exit 10 and the second exit 20 may each be configured fluidically connected to the first microfluidic channel 110 via the(ir respective) further connection channels 405. Hence, in embodiments, the further connection channels 405 may be configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from a microfluidic channel (e.g. 110) to the first and / or second exit 10,20.

[0180] In specific embodiments, the invention provides a method for the formation of artificial cell lipid bilayers. Especially, in embodiments, the method may comprise a first stage comprising providing organic solvent 1 comprising lipids to the first fluid inlet 111 of the microfluidic device 100. Subsequently, in embodiments, the first stage may comprise providing a flow of (first) aqueous phase 2 (optionally comprising microbeads) to the first fluid inlet 111 of the microfluidic device 100 to obtain an aqueous phase-organic phase interface 3 (between the organic solvent 1 and the (first) aqueous phase 2), such as depicted in Fig. 5. Further, in embodiments, the method may comprise a second stage comprising applying a pressure to the first fluid inlet 111 (at least) until the aqueous phase-organic phase interface 3 may be forced past the membrane formation section 200 via the n+1 elongated subchannels 110a, 110b, 110c. ... By forcing the aqueous phase-organic phase interface 3 past the membrane formation section 200 via the n+1 elongated subchannels 110a, 110b, 110c..., in embodiments, lipid bilayers (or membranes) 250 may be formed at the fluidic connection sections 105.

[0181] In specific embodiments, the method for the formation of artificial cell lipid bilayers may comprise the bubble reduction method. The invention may also provide a use of the microfluidic device 100 for (high- throughput) drug screening.

[0182] Furthermore, in embodiments, the invention provides a method for (high- throughput) active compound screening on artificial lipid bilayers. In embodiments, the method may comprise a first stage comprising providing lipid bilayers 250 in the microfluidic device 100 (by performing the above described method). Especially, in embodiments, the first stage may comprise after providing organic solvent 1 comprising lipids to the first fluid inlet 111, providing a flow of first aqueous phase 2 (optionally comprising microbeads) to the first fluid inlet 111 to obtain lipid bilayers 250 between the micropillars 210. Additionally, in embodiments, the first stage may comprise providing a flow of second aqueous phase 4 comprising an active compound to the second fluid inlet 121, and halting the flow of second aqueous phase 4 when one of the one or more connection channels 400 (especially the first connection channel 410) may be reached.

[0183] The method may, in embodiments, further comprise a second stage comprising after lipid bilayer formation blocking the second fluid outlet 122, halting the flow of first aqueous phase 2, and restarting the flow of second aqueous phase 4 to the microfluidic device 100 to provide the active compound to the formed lipid bilayers 250 (via the one or more connection channels 400.

[0184] Yet further, in embodiments, the method may comprise a third stage comprising analyzing the lipid bilayer 250 and / or the active compound (using optical characterization techniques) during the second stage and / or after the second stage.

[0185] The invention further provides a method for reducing the number of bubbles in a liquid (“bubble reduction method”). The bubble reduction method may, in embodiments such as depicted in Fig. 6, comprise providing the microfluidic device 100 and introducing a liquid 9 via the liquid inlet 101 in the microfluidic channel 109 and flowing the liquid 9 through one or more of the microfluidic channels 109. In this way the liquid 9 flows through the bubble trap section 300 and the one or more bubbles 5 may be trapped in the bubble trap section 300, especially providing a bubble-free liquid 5 downstream from the bubble trap section 300. The bubble-free liquid may, in embodiments, successively be directed to the membrane formation section 200 in the microfluidic device.

[0186] Hence, Fig. 6 schematically depicts an embodiment of the bubble reduction method. Moreover, the figure may depict an embodiment of the bubble reduction method comprised by method for the formation of artificial cell lipid bilayers and / or comprised by the method for (high-throughput) active compound screening on artificial lipid bilayers described above. In the figure a part of a microfluidic channel 109,110 comprising the bubble trap section

[0187] 300 is schematically depicted during carrying out the method at three successive moments in time. The initial moment is schematically depicted in microfluidic channel 109,110 indicated with the Roman I, the successive moment in time is schematically depicted in microfluidic channel 109,110 indicated with the Roman II, and the last moment in time is schematically depicted in microfluidic channel 109,110 indicated with the Roman III.

[0188] The figures schematically depicts that before the initial moment (I), an organic solvent 1 (e.g. comprising lipids) comprising microbeads was introduced in the microfluidic channel 109,110 (via the fluid inlet 101,111) and subsequently a flow of aqueous phase 2 was introduced. When changing from the organic solvent 1 supply to the supply of the aqueous phase 2, a bubble 5 may accidentally have been introduced in the microfluidic flow channel 109,110. At the initial moment (I) this results in the presence of the organic solvent 1 (configured furthest upstream), the bubble 5 (configured in the middle), and the aqueous phase 2 (configured furthest downstream), in the microfluidic channel 109, 110 at a location upstream of the bubble trap section 300.

[0189] After continuing the introduction of the aqueous phase 2, the organic solvent 1 flows into the bubble trap 300 through the first passage 301 and the second passage 302 and once the bubble 5 arrives at the bubble trap 300, the bubble 5 will flow into the first passage

[0190] 301 (having a larger primary flow through area than the second passage 302), whereas the aqueous phase 2 flows into the second passage 302. This is schematically indicated in the microfluidic channel indicated with II. Note that when comparing Fig. 6 with Fig. 1 and Fig 3, that the flow the passages 301,302 are configured differently.

[0191] A bit later in time, see the figure for the last moment in time (III), the aqueous phase 2 has overtaken the bubble 5 in the bubble trap 300. Successively, the bubble 5 may remain in the bubble trap section 300, and a bubble free liquid is provided at locations downstream of the bubble trap section 300.

[0192] In the depicted embodiment, a bubble is introduced between the provision of the organic solvent 1 and the provision of the aqueous phase 2. Such bubble 5 may easily be introduced when changing the supply to the microfluidic channel 109,110, and may e.g. also be introduced when changing between two aqueous phases 2 or between two organic solvents 1. Moreover also when providing a single liquid 9 to the microfluidic channel 109,110, the liquid 9 may comprise one or more bubbles 5 that preferably are removed. The bubble trap 300 may also be used for this kind (of combinations) of liquids. Having a single liquid 9 instead of different liquids 9 may further help removing bubbles 5. The bubble trap method may, in further embodiments, also be used for other liquids 9 than depicted in Fig. 6.

[0193] Furthermore, in embodiments, providing organic solvent 1 comprising lipids to the first inlet 111 of the microfluidic device 100 may comprise providing a volume X selected from the range of 0.3-100 pL of organic solvent 1 comprising lipids.

[0194] The invention may further provide a kit of parts for the formation of lipid bilayers. In embodiments, the kit of parts may comprise the microfluidic device 100, (one or more syringe pumps), a holder comprising an organic solvent 1 (and optionally comprising lipids), a holder comprising a first aqueous phase 2 (optionally comprising microbeads)(, and optionally a holder comprising a second aqueous phase 4).

[0195] As depicted Fig. 7, in embodiments, the invention further provides a system 1000 for performing optical measurements on artificial lipid bilayers. In embodiments, the system 1000 may comprise the microfluidic device 100. Moreover, in embodiments, the system 1000 may comprise a fluid managing device 50. The fluid managing device 50 may especially be fluidically connected with the microfluidic device 100. Especially, in embodiments, the fluid managing device 50 may be configured to provide fluids (such as one or more of the organic solvent 1, the first aqueous phase 2, and the second aqueous phase 4) to the first fluid inlet 111 and / or second fluid inlet 121 of the microfluidic device 100. Additionally or alternatively, in embodiments, the fluid managing device 50 may be configured to dispose of fluids exiting from one or more of the first fluid outlet 112, the second fluid outlet 122, the first exit 10, and the second exit 20. Furthermore, in embodiments, the system 1000 may comprise an optical measurement device 500. In embodiments, the optical measurement device 500 may be configured to provide radiation to the microfluidic device 100. Additionally or alternatively, in embodiments, the optical measurement device 500 may be configured to detect radiation emitted from the microfluidic device 100.

[0196] Experiments

[0197] Unless specified otherwise, the experiments described hereinafter were performed using the following materials and methods.

[0198] Fabrication of molds for the microfluidic device - Conventional lithography (etching SU-8) was implemented to make a master mold. To make a minor PDMS mold, the following steps were followed: PDMS and its curing agent (SYLGARD 184®) with 10: 1 ratio were well mixed, degassed in vacuum, and then casted on the master mold and degassed again. Afterwards it was cured in an oven at 85°C during 8 hours. Finally, the PDMS was diced out from the master mold and surface silanization (tri-chloro (lH,lH,2H,2H-perfluorooctyl)silane (PFOTS, Sigma-Aldrich)) was performed in a desiccator under vacuum. For the silanization, 50 pL of the silane in a glass tube was connected to the vacuumed chamber with a well-sealed connector for at least 2 hours such that the silane is vaporized and treats the surface of the mold.

[0199] Device fabrication protocol - The microfluidic device was fabricated by casting N0A81 (Norland Products) on the PDMS mold and placing the tip of a custom-made k-type thermocouple from Alumel and Chromel wires (TFAL-003 and TFCY-003, Omega) inside the liquid NOA. Then, a clean glass slide was deposited on top of the liquid NOA. The flow cell was then exposed to UV (Promed UVL-36 with four UV-9W-L bulbs) for 5 minutes. Afterwards, the PDMS minor mold was removed, inlet / outlet ports were drilled, and the channels were closed by bonding a cover slip previously spin-coated with N0A81 (partially UV-cured for 60 seconds). Finally, a 10-minute UV exposition was applied and the device was then baked for 8 hours on an 85 °C hot plate.

[0200] Lipids and membrane formation - l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), Palmitoyl-myristoyl-PC (PMPC), l,2-dipentadecanoyl-sn-glycero-3- phosphocholine, and l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) were procured from Avanti in chloroform at a concentration of 10 mg / ml. They were stored at -20 °C and used directly without additional purification. The first aqueous phase was prepared by adjusting a 10 mM 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) solution to pH 7.4 with concentrated KOH. Subsequently, KC1 (150 mM) and bovine serum albumin (BSA, 0.5 mg / ml) were added, and the aqueous buffer was filtered using a 0.2 pm sterile filter. To this, 2 pm beads (actual diameter = 1.93 pm, Polysciences Inc.) were diluted 25000 times in the aqueous buffer. The flow into the device was infused using a ProSense NE-30 syringe pump. While membrane formation was in progress, the pump was operated at high flow rates (5 pL / min) until all the chloroform was expelled from the device. Once the complete removal of chloroform was confirmed, the pump was stopped. Experiments were conducted on membranes aged between 2 to 8 hours.

[0201] Optical tweezers -All experiments were performed at 30 pm above the surface of the microfluidic device. Briefly, a 1.2 NA water immersion objective (FI Plan Apo VC 60*, Nikon) was used to optically trap a bead inside microchannels mounted on a piezostage (NANO-LPS100, Mad City Labs) with a 1064 nm trapping laser (YLR-10-LP-Y12, IPG Laser) and a 830 nm detection laser (LDT830-30GC, TOPAG). Antialiased filtering and preamplification (10 dB) was performed with a KROHN-HITE 3364 on the voltage signals from a position sensitive detector (PSD, DL100-7-PCBA3, First Sensor). In pushing experiments, fine positioning of the bead relative to the membrane was controlled with the piezo stage. The stage was moved sequentially in steps from 0.03 pm to 0.08 pm, and at each position, the data collection was done in a quasi-static manner by recording the PSD signal during 0.5 second, at a sampling rate of 1 kHz with a cut-off frequency of 500 Hz. For setup calibration, trap stiffness calculation, bubble trap quantification (flow measurement through the gap), and pushing experiments, signals were sampled at 50 kHz with a cut-off frequency of 24.5 kHz. All the signals from the PSD were processed and converted to nm and pN with a custom-made python code.

[0202] For the flow measurements to quantify the effect of the bubble trap, a similar process to the membrane formation procedure was followed. This time, chloroform, free of lipids, was injected into the microfluidic channels followed by 2 pm beads containing HEPES. After dynamic stabilization, the fluidic inlets remained connected to the pumping line and the fluidic outlets were not sealed. A 2 pm bead was trapped at the center of a gap while the pump was pushing at 2 pL / min and the signal was measured during 6 seconds.

[0203] Image processing for surface tension measurements - To track the membrane and the microbead, a CMOS camera (DCC1545M, Thorlabs GmbH) with 11.5 px / pm resolution was used to record videos of pushing experiments at 14 fps. Videos were converted to tiff format, cropped, and filtered with a FFT band pass filter for contrast enhancement in ImageJ. Modified tiff files were then analyzed with a custom Matlab script to obtain the deformation angle.

[0204] Statistical - A Kruskal-Wallis one-way statistical analysis was executed, utilizing a significance criterion of p = 0.05 to ascertain significance.

[0205] Video and image analysis for drug delivery test - For the laser induced fluorescent test (LIF), Rhodamine B was dissolved in HEPES 250 times and delivered at a rate of 2.5 pl / min to the microchip. LIF tests were captured with a 0.33 pixels / pm resolution and analyzed in ImageJ.

[0206] To track the beads for the observation of flow streamlines, TrackMate module of ImageJ was implemented. To preprocess the images, all the images were subtracted by the average image of all the stacks to remove the chip structures and the background noise. Then, the intensity was reversed to have high intensity for the beads and a dark background. Particle tracking tests were also filmed with a resolution of 0.33 pixels / pm.

[0207] Experiment 1 - Flow simulation

[0208] To achieve a precise fluidic and geometrical design for the microfluidic chip comprising (i) the first microfluidic channel comprising membrane formation section and bubble trap and (ii) the second microfluidic channel comprising the second bubble trap, it was necessary to use simulations to obtain a theoretically optimized design. Finite element method (FEM) was implemented to analyze and design the device in COMSOL Multiphysics 5.6. The designing procedure was conducted such that the microfluidic device might meet three criteria: The first criterion was to make sure that the flow at either side of any of the membranes remains symmetric during all the stages of the above described methods. For this purpose, the pressure must be balanced at all fluidic connection sections when membranes are formed and also when active compounds are delivered to them. The second criterion was to ensure that the concentration of the active compound to be delivered to the membranes was at the desired level and was also evenly distributed to all the membranes, including the membranes further from the first connection channel (i.e. further downstream the membrane formation section) since they are the last membranes to receive the molecule of interest. The third criterion was that the delivery / replacement should be done in a shear stress-free manner. This is crucial to be sure the membranes may not be damaged or their properties may not be affected by intense fluid shear stresses.

[0209] Taking these criteria into account, the laminar flow was modelled in the channels by solving the continuity and Navier-Stokes equations (in the two-dimensional domain of the device) in the steady-state utilizing the laminar flow module in COMSOL. This procedure is well-stablished in designing microfluidic systems. The first phase of simulation is when the pump connected to the first fluid inlet is run to form the bilayers. The important point here was that the flow should be symmetric at the fluidic connection sections so flat membranes may be formed without any warping.

[0210] With the optimized design, the COMSOL simulations showed that the first aqueous phase may reach the micropillars such that the flow and pressure on either side of the fluidic connection section may remain similar during this phase. Such results may specifically occur due to the streamline-shaped design of the membrane formation wall section making the geometry through which the fluid flows identical and symmetrical. Hence, three subflows of the flow partitioned by two rows of micropillars flowing in the membrane channel may behave similarly with less than 5% deviation (average flow rate of each subflow passing by micropillars). Additionally, the corresponding connection channels on either side of the first microfluidic channel may behave very similarly: the average flow rate going through each connection channel may be symmetric with less than 5% deviation. These two factors may cause parallel streamlines and balanced pressure distribution at the fluidic connection sections throughout the membrane formation section which may be favorable for the stable formation of flat membranes. The inlet flow rate during the simulation was 5 pL / min. After the membrane formation step, the flow of second aqueous phase was pumped into the second microfluidic channel. This simulation was done with a flow rate of 2.5 pl / min infusing into the device via the second fluid inlet. Although the device may also perform the delivery task at higher flowrates, it was experimentally found that 2.5 pl / min may be the maximum flowrate that formed membranes may tolerate without major warping or disruption, the simulation results showed that the streamline patterns at the fluidic connection sections may be parallel and a balanced pressure at all the gaps may be observed. The important point in the solvent replacement phase may be that magnitudes of fluid shear stress between the gaps may be negligible (at max 0.8 1 / s of shear rate). This may confirm that under these conditions, the replacement of solvents may be accomplished in an almost shear-free manner although convection may be the mechanism of delivery.

[0211] A simulation was performed to evaluate the “fallback delivery” scenario, which happens in case the first connection channel may be clogged and the second one may be used for replacement of the aqueous phase. Again, the flow may be favorable for membranes as it may be symmetric and balanced at all the fluidic connection sections.

[0212] Experiment 2 - Drug delivery (concentration) analysis

[0213] Simulations were also used to investigate whether the active compound at the second fluid inlet may reach all the membranes with a uniform concentration and also to find the time it may take for the second aqueous phase to fully replace the first aqueous phase. To do so, the laminar flow equations (continuity and Navier-Stokes) were coupled with the dilute mass transport equation and were solved in the two-dimensional domain for a time-dependent study in COMSOL, transport of dilute species module (with molecular species having a diffusion coefficient of D= 10'9m2 / s ). This simulation technique is well-used in drug-delivery screening microchips. Theoretically, in 230 seconds, both two last membranes may receive the same concentration of the delivered compound (with less than 3 percent error) as the other membranes upstream the flow. For the case of the using the second connection channel when the first connection channel may be out of circuit (fallback delivery), it may take 260 seconds for the membranes downstream the flow to experience a complete solvent replacement. The value of a convection-based replacement method shows itself here, where reversible solvent change may be done for dozens of membranes efficiently in less than 260 seconds.

[0214] Experiment 3 - Assessing flow while the replacement flow is pushed towards the artificial cell membranes

[0215] To experimentally observe the applicability of the microfluidic device in forming bilayers and in delivering the active compound (drug) to the membranes without disrupting them (i.e. maintaining the pressure balance at the fluidic connection sections during the replacement phase), a particle tracking experiment was performed. To do so, chloroform containing DOPC:DPPC (3:2) lipids was flown from the first fluid inlet into the microfluidic device, which was followed by a bead-free HEPES buffer (first aqueous phase). Consequently, membranes were formed at the fluidic connection sections between the micropillars and subsequent to the formation, the chloroform was absorbed into NOA81 until the annulus becomes undetectable. This happened within few seconds. At this point, some membranes may pop, but the rest of the membranes may maintain their planar stable configuration at the fluidic connection sections. Now, stable membranes may be at the fluidic connection sections while having a bead-free HEPES environment surrounding them. Next, the second aqueous solution, especially HEPES containing 2pm polystyrene beads diluted 3000 times from purchased solution (Polyscience Inc.) was flown into the device at the second fluid inlet at 2.5 pl / min. As predicted in simulations, the flow may go towards the membranes with streamlines parallel to the direction of the channel without adverse effects on the membranes. The particle flow around the first membrane (the membrane with the highest risk of flow asymmetry) was tracked in ImageJ to demonstrate that the flow symmetry was maintained during pumping the replacement flow.

[0216] Experiment 4 - Delivery of fluorescent dyes on the microfluidic device: Laser-induced fluorescent (LIE) test (LIE)

[0217] Although by tracking the beads, it was observed that they were delivered to the membranes and pressure was balanced, it was also vital to check experimentally if the concentration of the delivered molecules was as desired and stable over time. For this purpose, a Laser-induced fluorescent (LIF) test was conducted. Chloroform (no lipids) followed by the HEPES (no beads) where flown into the device from the first fluid inlet. When the first microfluidic channel was filled, Rhodamine B-containing HEPES was pushed from the second fluid inlet at the rate of 2.5 pl / min. The average concentration of the Rhodamine dye over time at the location of the last fluidic connection sections was recorded. Similar to the predictions of the mass transport simulation, within 210 seconds the concentration around the last fluidic connection sections may reach the input concentration (with less than 3% error). This may confirm that in 210 seconds the solvent may be fully replaced around all the membranes. At this stage, based on the need, the first aqueous phase may be reintroduced again or the experiments may be done with the second aqueous phase around membranes.

[0218] Experiment 5 - Indentation of an artificial cell membrane on the microfluidic device using optical tweezers For the measurement of membrane tension (<J) on the lipid bilayers, either a single-component (PMPC) or a binary-mixture (DOPC:DPPC(3:2)) composition was chosen to prove the capability of the microfluidic device on forming membranes with lipids of different lipid structure. For this purpose, the optical trap was used to push a 2 pm bead against a planar, freestanding lipid bilayer. The force-displacement curve of the trapped bead may provide a direct measurement of the surface tension and the subsequent calculation of the tube radius and bending rigidity. Qualitatively, for all lipid compositions in forward pushing, the initial portion of the curve may illustrate an approximately linear upward slope where the bead may gradually wrap within the bilayer. This trend may continue until it may reach a peak, signifying the forcebarrier to form the nanotube. The subsequent section may be a plateau, wherein a constant force may be required to extend the nanotube.

[0219] The membrane tension was calculated from the force data extracted from the force-displacement curves. The single-component composition (PMPC) shows a low tension value cfpMPc=3.52 ± 0.56 pN / m). The binary mixture composition, DOPC:DPPC(3:2), also forms stable membranes on the chip with the tension value of 6.75 ± 1 pN / m.The membrane tensions that were measured on the microchip were well in the range of other reports in the state-of-the-art using different platforms and measuring techniques.

[0220] Experiment 6 - Description and characterization of the bubble trap module

[0221] To quantify the effect of the bubble trap 300 on the pressure balance on each side of the lipid bilayers 250, the velocity of the flow around the fluidic connection sections 105 between the micropillars 210 was analyzed in a microfluidic device 100 without lipids 3 and the second microfluidic channel 120.

[0222] When the pressure is not balanced on either side of the fluidic connection sections 105, a flow can pass through the fluidic connection sections 105 which would prevent membrane formation or lead to the formation of curvy membranes in less extreme cases. With a balanced pressure, the flow on the midline of the fluidic connection sections 105 is expected to be parallel to the channel orientation, with no significant flow passing through the fluidic connection sections 105, favorizing membrane formation.

[0223] To measure the local flow velocities in the gap, a polystyrene bead of 2 pm diameter was optically trapped at the center of the fluidic connection sections 105, while liquid was flown through the first microfluidic channel 110 from the first fluid outlet 112 (for the case with no bubble trap 300) and from the first fluid inlet 111 (for the case with the bubble trap 300). For this purpose, a similar procedure as for lipid bilayer formation was followed, except that no lipid 5 was added to the organic solvent 1. Then, while a flow of 2 pL / min was applied with a syringe pump, the displacement of the microbeads from the center of the optical trap was measured for a 6-second period and converted to flow velocity for both cases (N = 3 experiments for each case). Without the bubble trap 300, the velocity along a y-axis (vy, of the microfluidic channel) reached 1216±153 pm / s, which indicated that fluid flows across the fluidic connection sections 105 because of a pressure imbalance between the subchannels in the membrane formation section 200 caused by the presence of bubbles 5 in the device 100. With the bubble trap 300 however, bubbles 5 were trapped upstream in the device 100 and no bubble 5 could reach the membrane formation section 200 to disturb the flow pattern. A significantly lower vyof 16±9 pm / s was found in the case of using a bubble trap 300, which demonstrated that the pressure in the subchannels in the membrane formation section was almost equal. This pressure balance shows the benefits of using the bubble trap 300 for membrane formation.

[0224] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0225] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0226] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0227] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

[0228] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0229] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0230] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0231] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0232] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0233] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0234] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0235] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0236] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system. The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0237] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A microfluidic device (100) for the formation of lipid bilayers, wherein the microfluidic device (100) comprises a first fluid inlet (111), a first fluid outlet (112), and a first microfluidic channel (110) configured to fluidically connect the first fluid inlet (111) and the first fluid outlet (112), wherein: the first microfluidic channel (110) comprises a channel wall (115) defining a channel height (He); wherein the first microfluidic channel (110) comprises a membrane formation section (200) having a membrane formation section axis (Emfs) of elongation, wherein the channel wall (115) at the membrane formation section (200) comprises a membrane formation wall section (215); the membrane formation section (200) comprises a plurality of micropillars (210), wherein each of the plurality of micropillars (210) is configured to extend along the channel height (He); the plurality of micropillars (210) are configured in an n*m array configured parallel to the membrane formation section axis (Emfs) of elongation; wherein the n*m array comprises n rows * m columns, wherein n is selected from the range of >1 and m is selected from the range of >2; wherein the n rows of micropillars (210) divide the first microfluidic channel (110) in the membrane formation section (200) into n+1 elongated subchannels (110a, 110b, 110c. . .); wherein the n*m array is configured to define per row of the n rows m-1 fluidic connection sections (105), wherein the m-1 fluidic connection sections (105) are configured between adjacent micropillars (21) and fluidically connecting adjacent elongated subchannels (110a, 110b, 110c...); the membrane formation section (200) is configured for forming lipid bilayers in the membrane formation section (200) in a direction parallel to the membrane formation section axis (Emfs) of elongation, wherein the lipid bilayers are formed at the fluidic connection sections (105); the plurality of micropillars (210) each have a maximum width (Wmax) defined in a direction perpendicular to the membrane formation section axis (Emfs) of elongation; and wherein each of the plurality of micropillars (210) tapers from its maximum width (Wmax) at least in directions parallel to the membrane formation section axis (Emfs) of elongation towards respective adjacent micropillars (210);the membrane formation wall section (215) is configured in a wave-like shape relative to the membrane formation section axis (Emfs) of elongation, wherein first distances (dl) defined between the membrane formation wall section (215) at the maximum width (Wmax) of the micropillars (210) and the membrane formation section axis (Emfs) of elongation, are smaller than second distances (d2) defined between the membrane formation wall section (215) at the fluidic connection sections (105) between the micropillars (210) and the membrane formation section axis (Emfs) of elongation.

2. The microfluidic device (100) according to claim 1, wherein n>2 and m>3.

3. The microfluidic device (100) according to any one of the preceding claims, wherein the membrane formation wall section (215) is configured in a wave-like shape mirroring the tapering of the plurality of micropillars (210).

4. The microfluidic device (100) according to any one of the preceding claims, wherein the microfluidic device (100) comprises a material selected from the group comprising: a glass, a polymeric material, and a cured optical adhesive.

5. The microfluidic device (100) according to any one of the preceding claims, further comprising a second fluid inlet (121), a second fluid outlet (122), a second microfluidic channel (120) configured to fluidically connect the second fluid inlet (121) and the second fluid outlet (122), and one or more connection channels (400), wherein the one or more connection channels (400) are configured to fluidically connect the second microfluidic channel (120) to the first microfluidic channel (110).

6. The microfluidic device (100) according to claim 5, wherein the one or more connection channels (400) each have: a total channel length (LRC) defined along a channel axis (CA) between (i) a first joint (401) of the respective connection channel (400) and the first microfluidic channel (110) and (ii) a second joint (402) of the respective connection channel (400) and the second microfluidic channel (120), wherein the total channel length (LRC) is selected from the range of 2-50 mm; and an equivalent cross-sectional circular diameter (DRC) defined perpendicular to the channel axis (CA) of the connection channel (400), wherein the equivalent cross-sectionalcircular diameter (DRC) is selected from the range of 50-500 gm, and wherein the connection channels 400 are configured to provide fluid control by providing a pressure-based resistance to the flow of fluids from one of the first microfluidic channel (110) and the second microfluidic channel (120) to the other one of the first microfluidic channel (110) and the second microfluidic channel (120).

7. The microfluidic device (100) according to any one of the preceding claims 5- 6, wherein the one or more connection channels (400) comprise a first connection channel (410) and a second connection channel (420); wherein the first connection channel (410) is configured upstream of the membrane formation section (200); wherein the second connection channel (420) is configured downstream of the membrane formation section (200); and wherein at least one of the one or more connection channels (400) is configured in a meandering pattern.

8. The microfluidic device (100) according to any one of the preceding claims, wherein the first microfluidic channel (110) further comprises a bubble trap section (300) configured downstream of the first fluid inlet (111) and upstream of the membrane formation section (200), wherein the optional second microfluidic channel (120) as defined in claim 5 also comprises a bubble trap section (300) configured downstream of the second fluid inlet (121) and upstream of the membrane formation section (200); wherein the bubble trap section (300) is configured to trap a gas bubble (5) in the bubble trap section (300).

9. The microfluidic device (100) according to claim 8, wherein the first microfluidic channel (110) comprises the bubble trap section (300) having a bubble trap section axis (EBT) of elongation, wherein the bubble trap section (300) is configured upstream of the membrane formation section (200), wherein the channel wall (115) at the bubble trap section (300) comprises a bubble trap wall section (315) extending along the channel height (He), wherein the bubble trap section (300) comprises a divider wall arrangement (310), wherein: the divider wall arrangement (310) is configured to divide the first microfluidic channel (110) in the bubble trap section (300) in a first passage (301) and a second passage (302); and the bubble trap wall section (315) and the divider wall arrangement (310) define (i) a primary first passage flow-through area (An) and a secondary first passage flow-through area (An) from the first passage (301), wherein the primary first passage flow-through area (An) is configured upstream of the secondary first passage flow-through area (An), and (ii) aprimary second passage flow-through area (A21) and a secondary second passage flow-through area (A22) from the second passage (301), wherein the primary second passage flow-through area (A21) is configured upstream of the secondary second passage flow-through area (A22); wherein An>A2i and An<A22.

10. Use of the microfluidic device according to any one of the claims 8-9 for the formation of lipid bilayers with a liquid (9), wherein upstream of the membrane formation section (200), gas bubbles (5) in the liquid (9) are trapped in the bubble trap section (300).

11. A method for the formation of artificial cell lipid bilayers, the method comprising:(i) a first stage comprising providing organic solvent (1) comprising lipids to the first fluid inlet (111) of the microfluidic device (100) according to any one of the preceding claims 1-9; and subsequently providing a flow of aqueous phase (2) to the first fluid inlet (111) of the microfluidic device (100) to obtain an aqueous phase-organic phase interface (3) between the organic solvent (1) and the aqueous phase (2); and(i) a second stage comprising applying a pressure to the first fluid inlet (111) until the aqueous phase-organic phase interface (3) is forced past the membrane formation section (200) via the n+1 elongated subchannels (110a, 110b, 110c. . .).

12. A method for active compound screening on artificial lipid bilayers, the method comprising:(i) a first stage comprising providing lipid bilayers (250) in the microfluidic device (100) according to any one of the preceding claims 1-9;(ii) a second stage comprising after lipid bilayer formation providing a flow of second aqueous phase (4) to the microfluidic device (100) to provide the active compound to the formed lipid bilayers; and(iii) a third stage comprising analyzing the lipid bilayer (250) and / or the active compound during the second stage and / or after the second stage.

13. Use of the microfluidic device (100) according to any one of the preceding claims 1-9 for drug screening.

14. A kit of parts for the formation of lipid bilayers, comprising:the microfluidic device (100) according to any one of the preceding claims 1-9; a holder comprising an organic solvent (1); and a holder comprising a first aqueous phase (2).

15. A system (1000) for performing optical measurements on artificial lipid bilayers, wherein the system (1000) comprises: the microfluidic device (100) according to any one of the preceding claims 1-9; a fluid managing device (50) configured to (i) provide fluids to the first fluid inlet (111) and / or second fluid inlet (121), as defined in claim 5, of the microfluidic device (100), and (ii) dispose of fluids exiting from one or more of the first fluid outlet (112), the second fluid outlet (122), a first exit (10), and a second exit (20); and an optical measurement device (500) configured to (i) provide radiation to the microfluidic device (100), and / or (ii) detect radiation emitted from the microfluidic device (100).

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