Microfluidic Devices

Abstract

A microfluidic device configured to sample, meter and recover a metered amount of a bodily fluid for analysis by capillary transport, the device comprising: an inlet section for receiving a sample of the bodily fluid, the inlet section including an inlet port and a channel system configured to transport the sample of the bodily fluid by stepwise or gradually increasing capillary action to a filtration membrane; a metering section configured to meter a predetermined amount of the received bodily fluid and separate it from the remainder of the fluid in the device; and an outlet section configured to receive and recover a metered amount of the bodily fluid from the metering section.

Description

[Technical field]

[0001] The present disclosure relates generally to microfluidic plasma extraction from whole blood and metering thereof, and more particularly to a microfluidic device configured to sample and recover a metered amount of bodily fluid for analysis by capillary transport, the device including a filtration membrane configured to separate selected cells from the bodily fluid and extract the bodily fluid. [Background technology]

[0002] Separation of plasma from whole blood is a key step in whole blood testing for clinical diagnostics and biomedical research purposes. Blood sampling is traditionally performed by venipuncture and collection of 5–10 ml of whole blood in a tube. For analysis, plasma is usually the desired material. Plasma is obtained by centrifugation in a centralized laboratory prior to analysis. As an alternative to working with liquid samples in tubes, blood can also be applied to a paper material and the sample allowed to dry on the paper. In the laboratory, the dried blood can be redissolved and prepared for analysis by wet chemistry. This method is called dried blood spot analysis (DBS) and, when combined with a separation technique that preserves blood cells, dried plasma spots (DPS) can also be obtained. This method has gained popularity as it offers the advantage of not having to maintain a cold chain during transport to the laboratory. The simple storage format also allows for capillary home sampling by finger prick.

[0003] Microfluidic systems and lab-on-a-chip are solutions that reduce the time and cost of biochemical assays. Miniaturization reduces the amount to be analyzed, shortens reaction times and especially reduces the consumption of expensive reagents. Microfluidic technology has also been applied for the purpose of plasma extraction. Separation of blood cells from plasma on the microscale can be achieved actively (externally applied forces such as electric or magnetic fields) or passively (sedimentation, filtration, hydrodynamic effects induced by microfeatures). Furthermore, paper-based as well as centrifugal microfluidics can also be applied.

[0004] For example, US 2014 / 0332098 A1 discloses circuit elements for self-powered, self-regulating microfluidic circuits, including programmable retention valves, programmable trigger valves, enhanced capillary pumps, and flow resonators. Some embodiments allow for reversing flow direction within the microfluidic circuit and for retaining reagents prior to sale or deployment of the microfluidic circuit, making it easier for users to use.

[0005] Many biochemical analyses require the quantification of analytes. Measuring the exact concentration of an analyte in a sample requires knowledge of the exact sample volume. At the microfluidic level, metering of liquids can again be achieved actively or passively. Examples of active means of dividing a liquid volume into two or more volumes include introducing components such as active valves that mechanically interfere with the liquid volume to divide it into units, or passive valves in combination with pressurized air that can tear apart parts of the liquid. In droplet microfluidics, the shear forces that arise between two immiscible liquid phases (oil and water) in certain microfluidic geometries (T-junctions) are utilized for liquid compartmentalization. Passive metering has not been widely reported in the literature. WO 2016 / 209147 A1 demonstrates passive using two dissolvable membranes integrated into a microchannel. Furthermore, US 2015 / 0147777 A1 uses a crossing overflow channel structure containing an absorbent material for metering. WO 2015 / 044454 A2 discloses a microfluidic device for collecting and transporting biological fluids, preferably whole blood, which includes a ramp and a metering channel for collecting a metered sample. The device has a first region of low flow resistance including an inlet feature and a second region including a metering channel with high flow resistance, an arrangement that can cause problems related to obtaining a stable performance to accommodate different flow rates due to variations in blood properties.

[0006] It is desirable to enable a fully autonomous system for plasma sampling. Such an autonomous system for plasma sampling has the advantage that it requires minimal interaction from the user to execute the process, thereby reducing the user's training level and reducing the risk of errors during sampling. An autonomous system by passive means at the microfluidic level can further reduce the complexity and cost of the system since no external driving force, such as a power source, is required to perform the microfluidic functions. However, developing such a system would involve substantial design challenges, such as making the system accommodating a wide range of whole blood characteristics in terms of the hematocrit, lipid content, and clotting factor variations that vary widely between individuals, as such variations create differences in flow characteristics within the system that are amenable to manipulation by active flow manipulation. The present disclosure is directed to improvements that solve the aforementioned problems, obtaining plasma samples with defined volumes.

[0007] One aspect of the problem to be addressed in microfluidic devices involves how to create height gradients in microfluidics, specifically in microfluidic substrates. Fabrication of microfluidic channels with gradient channel heights has been rarely performed in research or industrial microfluidic applications due to the difficulty of fabricating inclines or slopes on microfluidic substrates. The slopes may be fabricated by CNC micromilling, electroplating, or 3D printing. The resulting parts can be used as molds for, for example, injection molding or polymer casting. Unfortunately, these methods have limited resolution, resulting in the fabrication of stepped ladders rather than slopes, which is costly.

[0008] Height gradients play an important role in microfluidic systems. For example, He et al. increased the efficiency of a microfluidic mixer by 10% by sloping it. See Non-Patent Document 1. Microfluidic channels with trapezoidal cross sections have been applied in centrifugal microfluidics for particle separation (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). In these cases, the fabrication of such devices relied on complex and non-scalable fabrication protocols such as stereolithography.

[0009] Concentration gradients of chemicals or biomolecules in microenvironments play important roles in cell behaviors such as metastasis, embryonic development, axon guidance, and wound healing (Non-Patent Document 5). Because its size matches the scale of concentration gradients, microfluidics has become an efficient tool for manipulating fluid flow and diffusion profiles to create biomolecular gradients to study such cellular processes. Methods for generating concentration gradients generally utilize a diagonal branching configuration of rectangular microfluidic channels [Non-Patent Document 6]. Futai et al. fabricated a long-term concentration gradient generator by utilizing a height gradient in a microfluidic channel induced by manipulating photoexposed SU-8 resist to generate a slope in a PDMS mold [Non-Patent Document 7].

[0010] Lenk et al. (2006) showed that a plasma extraction membrane can be assembled at an angle in front of a microfluidic channel opening to form a wedge-like structure between the channel and the membrane to initiate capillary-driven plasma extraction. Hauser et al. (2006) show a similar device with a pinch-off structure for metered extracted plasma and a porous plug for collecting the plasma. WO 2020 / 050770 discloses a T-shaped configuration of the metering channel and a bridging element between the metering channel and the porous matrix. However, the T-shaped configuration has been found to be disadvantageous due to hematocrit dependency. Thus, these devices require improvement to accommodate changes in capillarity in the device to control or avoid the inclusion of air bubbles that may impair accuracy or reliable repeatable operation at various ranges of blood hematocrit values. Furthermore, improvements are required to comply with a simple and efficient large-scale production process. For example, WO 2011 / 003689 A2 discloses manufacturing issues with slopes for liquid transport. Unwanted bubble formation is a common problem in microfluidics. Choi et al. propose a solution using hydrophilic strips to overcome bubble formation when the fluid front flows from the channel into the large volume compartment. US 2009 / 0152187 discloses a plasma separation filter chip with a tapered shape toward the outlet to speed up the filtration process. However, no disclosure is made about the metering function or how to balance the capillarity at the inlet of the microfluidic device with plasma separation. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US Patent Publication No. 2014 / 0332098 [Patent Document 2] International Publication No. 2016 / 209147 Brochure [Patent Document 3] US Patent Publication No. 2015 / 0147777 [Patent Document 4] International Publication No. 2015 / 044454 Brochure [Patent Document 5] International Publication No. 2020 / 050770 Brochure [Patent Document 6] International Publication No. 2011 / 003689 Brochure [Patent Document 7] US Patent Publication No. 2009 / 0152187 [Non-patent literature]

[0012] [Non-Patent Document 1] Microfluidics and Nanofluidics volume 19, pages 829-836(2015) [Non-Patent Document 2] Scientific Reports volume 3, Article number: 1475 (2013) [Non-Patent Document 3] Micromachines (Basel). 2018 Apr; 9(4): 171 [Non-Patent Document 4] Scientific Reports volume 5, Article number: 7717 (2015) [Non-Patent Document 5] Electrophoresis 2010 Sep;31(18):3014-27 [Non-Patent Document 6] RSC Adv., 2017,7, 29966-29984 [Non-Patent Document 7] Micromachines (Basel)<https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6356992 / > 2019 Jan; 10(1): 9 [Non-Patent Document 8] Analytical chemistry 90 (22), 13393-13399 [Non-Patent Document 9] Analytical Chemistry 2019, 91, 7125-7130 Summary of the Invention [Problem to be solved by the invention]

[0013] It is an object of the present disclosure to provide an autonomous microfluidic capillary driven device having an inlet and metering section for metering and collecting sampled bodily fluids for analysis, the device having controlled capillary transport with a channel system that allows for increased capillary action.

[0014] It is an object of the present disclosure to provide an inlet section of a microfluidic device with controlled and increased capillarity for accessing a sample, such as blood, to a filtration membrane to support distribution over the filtration membrane surface to facilitate and control the extraction process of a filtered bodily fluid, such as plasma.

[0015] The objective of the present disclosure is to introduce functionality into microfluidic devices that allows them to accept sufficient bodily fluid volumes, which relies on simple observation and convenient user interaction to correct insufficient capacity.

[0016] The object of the present disclosure is to provide a capillary-driven device with a filtration membrane for the filtration of body fluids, which allows for precise separation of a well-defined volume of filtrate from the remaining fluid plug consisting of unfiltered and filtrate body fluids.

[0017] It is an object of the present disclosure to provide a device having a metering function that is capillary driven for filtration of bodily fluids and relies on an air-liquid interface with controlled air bubble introduction to support accurate transport and separation of metered fluids for collection.

[0018] It is also an object of the present disclosure to provide a microfluidic device that can filter and transport a blood sample, accurately meter a resulting plasma, and separate a metered plasma sample, and that operates reliably for all blood hematocrit levels.

[0019] It is also an object of the present disclosure to provide a microfluidic device that accepts a controlled injection volume of sample body fluid, correlated with the dead volume of the device, and a defined output volume is collected for analysis.

[0020] In the general aspects of this disclosure and below, the chambers and channels of the system are referred to as having carefully selected configurations to accurately transport, filter, meter and collect bodily fluids. Such configurations include dimensions of the chambers or channels designed to adequately support separation and collection of transported and metered volumes. Dimensions can be addressed in terms of the "height" or "width" of the chamber or channel. Other configurations can relate to the materials or other features that make up the chamber or channel, and in such contexts terms such as "floor" or "roof" are used. Thus, such terms have their usual meaning to those of skill in the art. In the context of this disclosure, microfluidic devices are configured with a "connector", "a fluid connector", or "a connecting piece". When these terms are used, they refer to connecting channels or chambers that are in fluid communication with adjacent parts of the device, are dimensioned as disclosed to support capillary transport within the device, and may introduce certain functionality into the device.

[0021] In a general aspect of the present disclosure, the term "capillarity" refers to the capillary pressure that exists at a liquid-air interface where surface or interfacial tension exists. Capillarity depends on the dimensions of the device, such as the pore size of the membrane, the type of liquid, such as aqueous or organic, salt content, etc., and the dimensions and / or surface properties of the flow channel, such as hydrophobicity or hydrophilicity, including the degree of hydrophobicity or hydrophilicity of the surface (contact angle). The terms "capillarity" and "capillary pressure" are both used in various contexts in the present disclosure. For example, the term "capillarity" is used functionally to describe device features such as channels and chambers. For example, the term "capillary pressure" is used in describing the implementation of the disclosed methods, such as transporting and metering bodily fluids through the device of the present disclosure. As referred to herein, "capillary means" refers to a porous member that can function as a capillary pump and collect bodily fluids for subsequent analysis of bodily fluid components.

[0022] The term "flow reduction means" in the context of this disclosure has a general meaning to characterize a channel or chamber of a device that temporarily reduces or stops the capillary flow of a bodily fluid from an inlet to an outlet of the device. Examples of flow reduction means include capillary stop valves, dissolvable valves, portions of a channel with altered hydrophilicity, portions of a channel with altered dimensions, portions of a channel with increased flow resistance, etc.

[0023] The term "pinch-off means" is generally used to describe the portion of the present disclosure where a predetermined volume of bodily fluid is separated from the remainder of the bodily fluid in the device. In this regard, the pinch-off is established by introducing an air bubble into an area of ​​the device where capillarity is low, a point where the resistance to air inlet is low compared to the surrounding areas. A "pinch-off means" according to the present disclosure can be used to reduce the flow resistance to introduce one or more air bubbles through one or more vents in the pinch-off area, and can be located in the pinch-off area designed to induce a low capillary pressure in the transported liquid column that can separate the metered volume of liquid from the remaining sampled volume by the device.

[0024] In the general aspect of the present disclosure and below, the term "capillary means" refers to a function that acts as a capillary pump and serves to collect a metered amount of body fluid in the device, optionally into a filtered body fluid, for subsequent analysis of one or an analyte. The skilled person will understand that the capillary means has a controlled porosity adapted to other parts of the device, as further explained in WO2015 / 044454. In the general aspect of the present disclosure and below, the term "body fluid" may relate to blood, and the filtered body fluid is plasma. It will be considered that other body fluids for transport, metering and collection can also be performed with the device. [Means for solving the problem]

[0025] In a first aspect of the present disclosure, there is provided a microfluidic device configured to sample, meter and recover a metered volume of a bodily fluid for analysis by capillary transport, the device comprising: an inlet section for receiving a sample of the bodily fluid, the inlet section including an inlet port and a channel system configured to transport the sample of the bodily fluid; a filtration membrane configured to separate plasma from blood; a metering section configured to meter and separate a predetermined volume of the received bodily fluid from the remainder of the bodily fluid within the device; and an outlet section configured to receive and recover a metered volume of the bodily fluid from the metering section, the outlet section including a capillary means for recovering the metered volume of the bodily fluid; The inlet section and channel system include a first channel, a second channel, and a third channel arranged in succession in a flow direction and in fluid communication with the inlet port, the inlet section and channel system configured to transport a sample of bodily fluid to and distribute across the filtration membrane in a stepwise or gradually increasing capillary action from the inlet section to the filtration membrane, the metering section includes an extraction chamber configured to receive bodily fluid extracted from the filtration membrane and arranged in fluid communication with the metering channel, and the metering section includes pinch-off means configured to separate the metered amount of bodily fluid, the pinch-off means including at least one vent hole arranged in a portion of the extraction chamber having a maximum height.

[0026] The step or gradual increase in capillarity ensures that the fluid sample is transported from the inlet section to the filtration membrane without retention, ensuring continuous operation of the device. Additionally, the step or gradual increase in capillarity allows for a nearly even distribution of filtration across the membrane. The vent achieves effective separation of the metered volume of fluid from the remaining volume of fluid.

[0027] In one embodiment, a stepwise or gradual increase in capillarity of the channel system is established by successively decreasing the height of the channels and / or successively increasing the hydrophilicity of the channels from the inlet port to the filtration membrane.

[0028] In one embodiment, the floor of the third channel is defined by the flat upper surface of the filtration membrane, and thus the third channel extends parallel to the filtration membrane to form a filtration chamber.

[0029] In one embodiment, the ratio of the height of the first channel to the second channel is at least 1.1:1, preferably at least 2:1, and the ratio of the height of the second channel to the third channel is at least 1.1:1, preferably at least 2:1, and preferably the height of the first channel is 500-2000 μm, the height of the second channel is 100-600 μm, and the height of the third channel is 25-200 μm.

[0030] In one embodiment, the second channel includes a capillary stop valve and a means for visual filling inspection, such as an inspection window, both located adjacent to the first channel outlet. The capillary stop valve allows the flow of bodily fluid through the channel system to be interrupted until the supply of bodily fluid is removed from the inlet port, whereby the capillary stop valve is breached by an increase in the Laplace pressure of the droplet formed at the inlet port overcoming the threshold pressure of the capillary stop valve. This can be used to meter the amount of bodily fluid before it flows into the second channel. A user can check the filling level with the visual inspection means to ensure that a sufficient amount has been delivered.

[0031] In one embodiment, the capillary stop valve is selected from at least one of a portion of the second channel having an altered hydrophilicity and / or a portion of the second channel having an altered dimension. The hydrophilicity and / or dimension of the second channel may be configured to achieve a desired threshold or breakthrough pressure of the capillary stop valve. Preferably, the capillary stop valve is formed by an abrupt increase in the height of the second channel.

[0032] In one embodiment, the pinch-off means comprises a pinch-off region arranged in fluid communication with one or more vents located in front of the inlet of the metering channel, the pinch-off region comprising a height reduction element having a height less than the maximum height of the brewing chamber, preferably with a through hole to prevent retention of liquid in the brewing chamber.

[0033] In one embodiment, the extraction chamber includes a portion of gradually increasing height, a portion having a height reducing element, and a portion having a maximum height disposed in fluid communication with the metering channel.

[0034] In one embodiment, the top of the extraction chamber is defined by the flat lower surface of the filtration membrane, and the floor of the extraction chamber extends at an acute angle from the contact point with the filtration membrane towards the metering channel. Preferably, the extraction chamber is generally wedge-shaped with a height that gradually increases from the contact point with the filtration membrane towards the metering channel, with the maximum height of the extraction chamber exceeding the height of the metering channel. The acute angle between the filtration membrane and the floor of the extraction chamber allows for a wedge-shaped extraction chamber that diverges (widens) towards the metering channel, thereby allowing the space between the diverging surfaces to be filled progressively, approximately forming a capillary pump. At the same time, the approximately flat and horizontal orientation of the filtration membrane can be maintained, facilitating the integration of the filtration membrane into the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction.

[0035] In one embodiment, the first channel has a volume that correlates to the dead volume of the device and the metered volume (output volume). Preferably, the volume of the first channel is sufficient to prevent the front meniscus of a body fluid volume other than the metered volume from reaching the capillary means of the outlet section. The dead volume is the sum of all volumes that are not metered and are collected by the capillary means of the outlet section. In other words, the dead volume is the residual volume in the system that is distributed across the filtration chamber, the plasma extraction (filtration) membrane, and the plasma extraction chamber. The plasma output (metered) volume is the volume that is separated from the dead volume, for example by the pinch-off effect. Since the input volume applied by the user of the device to the inlet port varies and the metered output volume is constant and predetermined by the device, the dead volume also varies within an acceptable range. Thus, the volume of the first channel correlates to the dead volume and the output metered volume. By selecting the volume of the first channel in this way, it is ensured that only the amount of blood required for plasma sampling flows into the first channel.

[0036] In one embodiment, the metering channel has an outlet portion having a dimensional change configured to cause a fluid front meniscus of the separated metered amount of bodily fluid to assume a shape that generally matches the surface shape of the capillary means as it is transported to the outlet section. The dimensional change in the outlet portion of the metering channel can cause the shape of the fluid front meniscus to conform to the shape of the capillary means such that they match each other at the interface. This can control the impingement of the separated metered amount of bodily fluid onto the capillary means to prevent bubble formation between the two media.

[0037] In one embodiment, the dimensional change includes a decrease in the width and / or height of the metering channel, which can overcome any effects of surface roughness or dimensional variations in the metering channel to induce the formation of a substantially straight or planar meniscus.

[0038] In one embodiment, the distal end of the outlet portion of the metering channel adjacent the capillary means has a constant width less than the width of the metering channel. Preferably, the outlet portion of the metering channel has a first portion that gradually decreases in width and a second portion that has a constant width less than the width of the metering channel. The reduction in width causes the fluid meniscus to go from a convex shape to a generally planar shape that matches the shape of the capillary means.

[0039] In one embodiment, the surface profile of the capillary means at the interface with the fluid front meniscus is curved or approximately planar.

[0040] In one embodiment, the outlet section comprises a hydrophilic porous bridge element having an average pore size smaller than the smallest dimension of the metering channel, the bridge element being disposed in fluid communication with the outlet portion of the metering channel and the capillary means, the provision of the capillary means in two components enhances capillary action to ensure transport of a metered volume of separated bodily fluid from the metering channel to the paper substrate for collection.

[0041] Further, a first aspect of the present disclosure relates to a method for sampling, transporting and recovering a metered volume of a bodily fluid for analysis by capillary transport in a microfluidic device, the method comprising the steps of: providing the bodily fluid to an inlet port of the device; filling a channel system arranged in fluid communication with the inlet port, the channel system comprising, successively in the direction of flow, a first channel, a second channel and a third channel arranged in fluid communication with the inlet port; and separating the sample of the bodily fluid and plasma from blood by stepwise or gradually increasing capillary action. the bodily fluid sample being transferred to a filtration membrane configured to filter the bodily fluid; distributing the sample of the bodily fluid across the filtration membrane; receiving the filtered bodily fluid in a metering section including an extraction chamber and a metering channel in fluid communication with the extraction chamber; transporting the filtered bodily fluid in the metering channel to an outlet section including a capillary means for collecting the filtered bodily fluid; isolating the metered amount of filtered bodily fluid by introducing at least one gas bubble into a portion of the metering section that induces a minimum capillary pressure; and collecting the metered amount of filtered bodily fluid in the capillary means.

[0042] In one embodiment the method is carried out using a device according to the first aspect, using a blood sample from which plasma is measured and collected.

[0043] In a second aspect of the present disclosure, there is provided a microfluidic device configured to sample, meter and recover a metered volume of a bodily fluid for analysis by capillary transport, the device comprising: an inlet section for receiving a sample of the bodily fluid, the inlet section including an inlet port and a channel system; a filtration membrane configured to separate plasma from blood, the inlet port and the channel system configured to transport and distribute the sample of the bodily fluid across the filtration membrane with a stepwise or gradually increasing capillarity from the inlet port to the filtration membrane; a metering function configured to meter a predetermined amount of the received bodily fluid; and at least one porous medium for receiving the transported sample of the bodily fluid.

[0044] The step or gradual increase in capillarity ensures that the sample of bodily fluid is transported from the inlet section to the filtration membrane without pinning, ensuring continuous operation of the device, and furthermore, the step or gradual increase in capillarity can be distributed across the membrane so that filtration occurs approximately evenly across the membrane.

[0045] In one embodiment, the channel system comprises at least two flow paths, including a first channel disposed in fluid communication with the inlet port and a second channel having higher capillarity than the first channel. In one embodiment, the ratio of the height of the first channel to the second channel is at least 1.1:1, preferably at least 2:1. With at least two flow paths, the increase in capillarity can be achieved in at least two stages, for example through a decrease in height.

[0046] In one embodiment, the channel system comprises at least one of a flow reduction means and a visual filling inspection means, such as an inspection window. Preferably, the filling inspection means is provided in a second channel adjacent to the first channel. The flow reduction means and the filling inspection means allow pre-metering by interrupting the flow of the sample so that the operator can stop applying the body fluid to the device when a sufficient amount has been added, i.e. when the channel system is filled.

[0047] In one embodiment, the flow reduction means is selected from at least one of a portion of the second channel having altered hydrophilicity, a portion of the second channel having altered dimensions, and a portion of the second channel having increased flow resistance, preferably the flow reduction means is provided adjacent to the means for visual inspection. Preferably, the flow reduction means is a dissolvable valve or a capillary stop valve, preferably the capillary stop valve comprises an abrupt increase in the height of the second channel.

[0048] In one embodiment, the porous medium is configured to absorb and withdraw the received volume, preferably the porous fluid medium is a lateral fluid medium or filter paper.

[0049] In one embodiment, the metering function comprises a metering section configured to receive bodily fluid extracted from the filtration membrane and having an extraction chamber disposed in fluid communication with the metering channel, and the device further includes an outlet section configured to receive and collect a metered amount of bodily fluid from the metering channel, the outlet section including a capillary means for collecting the metered amount.

[0050] In one embodiment, the channel system comprises a first channel having a first capillary action and disposed in fluid communication with the inlet port, and a third channel having a second capillary action, the second capillary action being higher than the first capillary action, the third channel including a top portion, optionally a vent, and configured to distribute a sample of bodily fluid arriving from the first channel uniformly across the filtration membrane. Preferably, the third channel comprises a floor defined by a flat upper surface of the filtration membrane.

[0051] In one embodiment, a stepwise or gradual increase in capillarity of the channel system is established by successively decreasing the height of the channels and / or increasing the hydrophilicity of the channels from the inlet port to the filtration membrane. Preferably, the stepwise increase in capillarity of the channel system from the inlet port to the filtration membrane is established over at least two steps.

[0052] In one embodiment, the first channel has a volume that correlates to the dead volume and the metered volume of the device, and preferably the volume of the first channel is sufficient to prevent the front meniscus of the body fluid volume other than the metered volume from reaching the capillary means of the outlet section. The dead volume is the sum of all volumes that are not metered and are collected in the capillary means at the outlet. In other words, the dead volume is the residual volume in the system distributed across the filtration chamber, the plasma extraction (filtration) membrane, and the plasma extraction chamber. The plasma output (metered) volume is the volume that is separated from the dead volume, for example by the pinch-off effect. Since the input volume applied to the inlet port by the user of the device varies, but the metered output volume is constant and predetermined by the device, the dead volume also varies within an acceptable range. Thus, the volume of the first channel correlates to the dead volume and the output metered volume. This selection of the volume of the first channel ensures that only the amount of blood required for plasma sampling flows into the first channel.

[0053] In one embodiment, the device further comprises a second channel disposed between and in fluid communication with the first and third channels. The second channel provides an additional step in the channel system to achieve a stepwise or gradual increase in capillarity. Preferably, the height ratio of the second channel to the third channel is at least 1.1:1, preferably at least 2:1.

[0054] In one embodiment, the extraction chamber is generally wedge-shaped with a gradually increasing height from contact with the filtration membrane towards the metering channel, the maximum height of the extraction chamber being greater than the height of the metering channel. The wedge shape allows for gradual filling of the extraction chamber.

[0055] In one embodiment, the device further comprises a pinch-off means configured to separate the metered amount of bodily fluid, the pinch-off means including at least one vent hole located in the portion of the extraction chamber having the maximum height, the vent hole providing an effective separation of the metered amount from the remaining amount of bodily fluid.

[0056] In one embodiment, the pinch-off means comprises a pinch-off region in fluid communication with at least one vent hole disposed adjacent to the inlet of the metering channel, the pinch-off region comprising a height reduction element having a height less than the maximum height of the extraction chamber. Preferably, the extraction chamber comprises a portion with a gradually increasing height, a portion with a height reduction element, and a portion with the maximum height of the extraction chamber in fluid communication with the metering channel. The height reduction element increases capillarity at the outlet of the extraction chamber, thus ensuring continuous transport and filtration of the body fluid through the filtration membrane.

[0057] In one embodiment, the height reducing element includes through holes to prevent liquid retention.

[0058] Furthermore, a second aspect of the present disclosure relates to a method for sampling, metering and recovering a body fluid sample for analysis by a microfluidic device as embodied in this second aspect, comprising the steps of injecting a sample volume into an inlet port of the device and transporting the sample volume to a porous filtration membrane through a channel system that allows a continuous increase in capillary pressure, preferably a stepwise increase in capillary pressure. The method further comprises the steps of allowing further increased capillary pressure from the porous filtration membrane to separate cellular material and extract the remaining body fluid, receiving the filtered body fluid from the filtration membrane into an extraction chamber that induces gradually lower capillary pressures, filling the metering channel with the filtered body fluid by the increased capillary pressure, blocking the fluid communication between the extraction chamber and the metering channel by introducing an air bubble at a predefined time point such that the body fluid experiences the lowest capillary pressure, and recovering the metered body fluid in a capillary means included in the outlet section. Preferably, when the metered body fluid contacts the capillary means, fluid communication between the extraction chamber and the metering channel is interrupted.

[0059] In an embodiment of the method, a volume of bodily fluid is manually applied to the inlet port, through which the fluid is introduced to fill the first channel, and once the first channel is filled, a flow reduction means temporarily stops or reduces transport of the bodily fluid. After verifying that the device is properly filled, excess bodily fluid is removed from the inlet port, and further transport requires a separation, metering, and recovery procedure.

[0060] In a third aspect of the present disclosure, there is provided a microfluidic device configured to sample, meter and collect a metered amount of bodily fluid for analysis by capillary transport, the device comprising: an inlet section including an inlet port for receiving a sample of bodily fluid; a metering section configured to receive the bodily fluid from the inlet section and comprising a metering channel, the metering section being arranged to separate a metered volume of the bodily fluid loaded into the metering channel; and an outlet section configured to receive and transport the separated metered amount of bodily fluid for collection into a capillary means having a predetermined surface shape, the outlet section having an outlet portion having a dimensional change configured such that a fluid front meniscus of the separated metered amount of bodily fluid assumes a shape that generally matches the surface shape of the capillary means when transported into the outlet section.

[0061] The dimensional change of the outlet portion of the metering channel allows the shape of the fluid front meniscus to be adapted to the shape of the capillary means such that the shapes of the interfaces match each other, thereby controlling the impingement of the metered amount of separated body fluid onto the capillary means and preventing bubble formation between the two media.

[0062] In one embodiment, the dimensional change includes a reduction in the width and / or height of the metering channel, which can overcome any effects of surface roughness or dimensional variations in the metering channel to cause the formation of a substantially straight or planar meniscus.

[0063] In one embodiment, the distal end of the outlet portion of the metering channel adjacent the capillary means has a constant width less than the width of the metering channel. Preferably, the outlet portion of the metering channel has a first portion that gradually decreases in width and a second portion that has a constant width less than the width of the metering channel. This decrease in width causes the fluid meniscus to transition from a convex shape to a generally planar shape that matches the shape of the capillary means.

[0064] In one embodiment, the surface shape of the capillary means at the interface with the fluid front meniscus is curved or approximately planar.

[0065] In one embodiment, the capillary means includes a bridge element disposed in fluid communication with the outlet portion of the metering channel and a paper substrate connected to the bridge element. Preferably, the bridge element is a hydrophilic porous element having an average pore size smaller than the smallest dimension of the metering channel. By providing the capillary means in two components, increased capillary action can be achieved to ensure transport of the separated metered volume of bodily fluid from the metering channel to the paper substrate for collection.

[0066] In one embodiment, the bridging element is made from a material selected from at least one of micropaper pulp, microfibrillated cellulose, an open cell hydrophilic polymer, or a highly compressible glass fiber web.

[0067] In one embodiment, the surface profile of the bridge element at the interface surface with the fluid front meniscus is curved or substantially planar.

[0068] In one embodiment, the device further includes a filtration membrane configured to separate selected cells from the bodily fluid, the inlet section configured to transport a sample of the bodily fluid to the filtration membrane and distribute the sample of the bodily fluid across the filtration membrane, and the metering section includes an extraction chamber configured to receive the bodily fluid from the filtration membrane and transport the received bodily fluid to the metering channel. The filtration membrane can separate plasma or the like from whole blood collected by capillary means.

[0069] In one embodiment, the device further comprises a pinch-off means configured to separate the metered amount of bodily fluid, the pinch-off means including at least one vent hole located in the portion of the extraction chamber having the maximum height, the vent hole providing an effective separation of the metered amount of bodily fluid from the remaining amount of bodily fluid.

[0070] In one embodiment, the pinch-off means comprises a pinch-off area in fluid communication with the at least one vent hole, the pinch-off area being located in a portion of the extraction chamber having a maximum height and being surrounded by an area having a lower height. Preferably, at least a portion of the extraction chamber surrounding the pinch-off area has a height that is lower than the height of the metering channel. The surrounding area of ​​reduced height leads to a reduction in the capillary pressure in the pinch-off area, facilitating the introduction of air bubbles.

[0071] In one embodiment, the metering section includes a fluid connector extending between the extraction chamber and the metering channel, and an air vent. The air vent may be located adjacent to the fluid connector or at a location where the fluid connector abuts the metering channel. Preferably, the air vent is located at the entrance to the metering channel and configured as an orifice to ambient air having a cross-sectional area equal to or greater than the cross-sectional area of ​​the metering channel. The air vent is thus located in a location of the device with low capillary pressure that is optimal for introducing air bubbles downstream of the extraction chamber and upstream of the metering channel to separate the metered volume of bodily fluid.

[0072] In one embodiment, the fluid connector has a different dimension than the metering channel, the dimension being selected from one or more of height, width and length. Preferably, the fluid connector has a height that gradually increases toward the inlet of the metering channel, thereby increasing the fluid / air interface and facilitating the introduction of air bubbles.

[0073] In one embodiment, the maximum height of the extraction chamber is less than the height of the metering channel.

[0074] Furthermore, a third aspect of the present disclosure relates to a method for sampling, transporting and recovering a metered volume of bodily fluid for analysis by capillary transport from an inlet of a microfluidic device to a capillary means, the method comprising the steps of injecting a sample of the bodily fluid into an inlet port of the device and transporting the bodily fluid, optionally through a filtration membrane, to a metering channel; allowing the metering channel to transport the sample of the bodily fluid to an outlet section comprising a capillary means having a predetermined surface shape; receiving the metered fluid within the capillary means and separating the metered volume of bodily fluid from the remaining sample volume by introducing at least one air bubble into a portion of the device upstream of the metering channel indicating low capillary pressure; and recovering the metered volume of bodily fluid into the capillary means, wherein the outlet portion of the metering channel configures a dimensional change that causes a fluid front meniscus of the separated volume of bodily fluid to assume a shape that approximately matches the surface shape of the capillary means as it is transported to the outlet section.

[0075] In a fourth aspect of the present disclosure, there is provided a method for manufacturing an outlet section of a microfluidic device configured to sample, meter and recover a metered amount of a bodily fluid for analysis by capillary transport, the method comprising the steps of: preparing a microfluidic device having an outlet section in fluid communication with a metering section including a metering channel configured to receive a bodily fluid from an inlet section having an inlet port, the outlet section including a bridge cavity between an outlet portion of the metering channel and an outlet orifice of the device; preparing a hydrophilic porous bridge element arranged to conform to a shape of the bridge cavity; inserting the bridge element into the bridge cavity such that the bridge element substantially fills the bridge cavity and the outlet orifice; and attaching a capillary means to the outlet section, thereby establishing contact between the capillary means and the bridge element.

[0076] By inserting a conformable hydrophilic porous bridge element into the bridge cavity so that the bridge cavity is nearly filled, the need for precision cutting and placement of the porous element at the outlet is reduced or eliminated. Instead, the method according to the fourth aspect allows for application of the solution in automated high throughput mass manufacturing.

[0077] In one embodiment, the insertion causes the bridge element to protrude into the metering channel. Preferably, the insertion causes a surface of the portion of the bridge element that protrudes into the metering channel to assume a shape that approximately matches the fluid front meniscus of a metered amount (volume) of bodily fluid in the metering channel. Thus, impingement of the metered amount of separated bodily fluid onto the bridge element can be controlled to prevent bubble formation between the two media.

[0078] In one embodiment, the bridge element is made of a compressible porous material and has a volume greater than the volume of the bridge cavity, and inserting comprises compressing the bridge element into the bridge cavity. Using a compressible material ensures that no gaps are formed between the bridge cavity and the bridge element upon simple compressive insertion of the bridge element into the bridge cavity.

[0079] In one embodiment, the bridge element is made of a dispenseable porous material, and inserting comprises dispensing the porous material into the bridge cavity such that it protrudes outside the exit orifice and allowing the porous material to solidify to form the bridge element. The use of a dispenseable material ensures that simply dispensing the bridge element into the bridge cavity does not form a gap between the bridge cavity and the bridge element. In this context, a dispenseable material encompasses any suitable material in liquid form that is dispensed into the bridge cavity, for example through a nozzle, and then hardened or solidified.

[0080] In one embodiment, the capillary means is configured to exert a higher capillary pressure on the body fluid than the bridge element, the bridge element having an average pore size smaller than the smallest dimension of the metering channel, thereby ensuring that the body fluid sample is transported from the metering channel through the bridge element to the capillary means.

[0081] In one embodiment, the bridging element is made from a material selected from at least one of micropaper pulp, microfibrillated cellulose, an open cell hydrophilic polymer, or a highly compressible glass fiber web.

[0082] Furthermore, a fourth aspect relates to a microfluidic device configured to sample, meter and recover a metered amount of bodily fluid for analysis by capillary transport, the device comprising: an inlet section including an inlet port for receiving a bodily fluid sample; a metering section configured to receive the bodily fluid from the inlet section and including a metering channel, the metering section being arranged to separate a metered amount of the bodily fluid which is filled into the metering channel; and an outlet section including a bridge cavity between an outlet portion of the metering channel and an outlet orifice of the device; a hydrophilic porous bridge element arranged to conform to a shape of the bridge cavity and inserted into the bridge cavity so as to substantially fill the bridge cavity and the outlet orifice; and a capillary means attached to the outlet section to contact the bridge element.

[0083] In one embodiment, the device further comprises a filtration membrane configured to separate selected cells from the bodily fluid, the inlet section configured to transport and distribute a sample of the bodily fluid across the filtration membrane, and the metering section includes an extraction chamber configured to receive the bodily fluid from the filtration membrane and transport the received bodily fluid to the metering channel. The filtration membrane can separate plasma or the like from whole blood collected in the capillary.

[0084] In one embodiment, the metering section includes a fluid connector extending between the extraction chamber and the metering channel, and an air vent. The air vent may be located adjacent to where the fluid connector abuts the metering channel, or may be located at a location where the air vent abuts the metering channel. In this manner, the air vent is located at a location of low capillary pressure in the device and optimally positioned to introduce air bubbles downstream of the extraction chamber and upstream of the metering channel to separate a metered amount of bodily fluid. Preferably, the fluid connector has a different dimension than the metering channel, the dimension being selected from one or more of height, width, and length.

[0085] In one embodiment, the outlet portion of the metering channel is configured such that the fluid front meniscus of the metered amount of separated bodily fluid assumes a shape that approximately matches the surface shape of the capillary means when transported to the outlet section. Preferably, the surface of the bridge element facing the metering channel is curved or approximately planar. Thus, the impact of the metered amount of separated bodily fluid on the bridge element can be controlled to prevent air bubbles from forming between the two media.

[0086] In one embodiment, the device further comprises a pinch-off means configured to separate the metered amount of bodily fluid, the pinch-off means comprising at least one vent hole disposed in the portion of the extraction chamber having the maximum height, the vent hole providing an effective separation of the metered volume from the remaining amount of bodily fluid.

[0087] In one embodiment, the pinch-off means comprises a pinch-off area in fluid communication with at least one vent hole, the pinch-off area being located in the part of the extraction chamber having the greatest height and surrounded by an area having a lower height. Preferably, at least a part of the extraction chamber surrounding the pinch-off area has a height lower than the height of the metering channel. The surrounding area of ​​reduced height leads to a reduction in the capillary pressure in the pinch-off area, thus facilitating the introduction of air bubbles.

[0088] In one embodiment, the maximum height of the extraction chamber is less than the height of the metering channel.

[0089] In one embodiment, the extraction chamber is approximately wedge-shaped, the top of the extraction chamber is defined by the flat lower surface of the filtration membrane, and the hydrophilic floor of the extraction chamber extends from the contact with the filtration membrane toward the metering channel at an acute angle. The acute angle between the filtration membrane and the floor of the extraction chamber allows the wedge-shaped extraction chamber to be realized, which diverges toward the metering channel, thereby allowing the space between the diverging surfaces to be gradually filled, essentially forming a capillary pump. At the same time, the approximately flat and horizontal orientation of the filtration membrane can be maintained, which facilitates the integration of the filtration membrane in the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction. Preferably, the hydrophilic floor is the floor of a fluid connector extending between the extraction chamber and the metering channel.

[0090] In one embodiment, the fluid connector has a maximum height and a minimum height that is less than the maximum height of the extraction chamber.

[0091] In a fifth aspect of the present disclosure, there is provided a multi-layer microfluidic device configured to sample, meter and recover a metered amount of a bodily fluid for analysis by capillary transport, the device comprising: an inlet section for receiving a bodily fluid sample, the inlet section including an inlet port and configured to transport and access the sample to a flat, laterally extending filtration membrane; a metering section comprising an extraction chamber and a metering chamber configured to receive bodily fluid extracted from the filtration membrane and disposed in fluid communication with the metering channel; and an outlet section configured to receive and recover a metered amount of bodily fluid from the metering flow path, the outlet section comprising a capillary means for recovering the metered volume of bodily fluid, a top of the extraction chamber defined by a flat lower surface of the filtration membrane, a floor of the extraction chamber contiguous with and extending at an acute angle from the lower surface of the filtration membrane, the floor of the extraction chamber being inclined relative to the floor of the metering channel to form a gradient.

[0092] By sloping the floor of the extraction chamber, a wedge-shaped extraction chamber diverging towards the metering channel can be achieved, thereby allowing the space between the diverging surfaces to fill gradually, creating an almost capillary pump, while at the same time maintaining a nearly flat and horizontal orientation of the filtration membrane, facilitating its integration in the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction.

[0093] In one embodiment, the device includes, from bottom to top, a bottom layer, a hydrophilic bed layer forming the floor of the extraction chamber and the metering channel, and a support structure for the bed layer, a first portion of the bed layer supported on the support structure and disposed between the bottom layer and the bed layer in contact with the filtration membrane, and a second portion of the bed layer supported on the bottom layer, forming an acute angle between the filtration membrane and the bed layer to obtain an extraction chamber with a gradually increasing height toward the metering channel. The layer structure allows for easy assembly of the device and scalable mass production.

[0094] In one embodiment, the apparatus includes at least five layers selected from a bottom layer, a support structure, a bed layer, a channel structure layer configured to accommodate the metering section, and a cover layer that provides a flat top surface for the metering channel.

[0095] In one embodiment, the bed layer includes a slot that defines a tongue that forms the floor of the extraction chamber, the free end of the tongue being supported on a support structure. Preferably, the slot is substantially C-shaped and the tongue is substantially circular or substantially square. The slot allows the tongue that forms the floor of the extraction chamber to be easily cut to the desired shape, for example to match the shape of the filtration membrane.

[0096] In one embodiment, the bed layer includes an opening that forms an outlet port of the outlet section.

[0097] In one embodiment, the bottom layer includes a first opening that corresponds approximately to the size of the extraction chamber and a second opening that is positioned to receive the capillary means.

[0098] In one embodiment, the channel structure layer includes openings arranged to accommodate the support structure, the floor of the extraction chamber and the outlet port of the outlet section, and preferably said channel structure layer further includes slots forming the sidewalls of the metering channels.

[0099] In one embodiment, the cover layer includes an opening that approximately corresponds to the size of the extraction chamber, with the lower surface of the filtration membrane positioned over the opening.

[0100] The openings in the different layers accommodate different structures forming a microfluidic device, allowing for multi-layer structures.

[0101] In one embodiment, the cover layer has a first side facing the channel layer having a hydrophilic surface and a second, opposing side surface having an adhesive surface, such that the hydrophilic surface forms the top of the metering channel and the adhesive surface allows for the assembly of additional layers on top of the cover layer.

[0102] In one embodiment, the device further includes at least one additional layer attached to the second surface of the cover layer for assembling the inlet section and the device housing.

[0103] Furthermore, a fifth aspect of the present disclosure relates to a method for manufacturing a microfluidic device by lamination of foil layers, the method comprising the steps of: preparing a substrate as a bottom layer of the device; assembling a support structure on the bottom layer; preparing a bed layer having a hydrophilic upper surface, the bed layer being assembled on the bottom layer such that a first portion of the bed layer is supported on the support structure and a second portion of the bed layer is supported on the bottom layer, the first portion of the bed layer being inclined relative to the second portion to form a gradient; preparing a channel structure layer configured to accommodate a metering section, the channel structure layer being assembled on the channel bed layer; preparing a cover layer and assembling the cover layer on the channel structure layer; and assembling a filtration membrane horizontally to rest on the cover layer, thereby forming an extraction chamber with the first portion of the bed layer as the floor.

[0104] The present fabrication method enables scalable mass production of multi-layer microfluidic devices with wedge-shaped extraction chambers.

[0105] In one embodiment, the method further includes forming a slot in the floor layer that defines a tongue forming the first portion, and assembling the floor layer on the bottom layer such that a free end of the tongue is supported on a support structure.

[0106] In one embodiment, the bed layer includes an opening that forms an outlet port of the outlet section.

[0107] In one embodiment, the bottom layer includes a first opening that corresponds approximately to the size of the extraction chamber and a second opening that is positioned to receive the capillary means.

[0108] In one embodiment, the channel structure layer includes an opening positioned to accommodate the support structure, the floor of the extraction chamber, and the outlet port of the outlet section.

[0109] In one embodiment, the cover layer has a first side facing the channel structure layer having a hydrophilic surface and a second, opposing side having an adhesive surface.

[0110] In one embodiment, the method further comprises assembling at least one additional layer onto the cover layer, and subsequently assembling the inlet section and the housing onto the at least one additional layer.

[0111] In a sixth aspect of the present disclosure, there is provided a microfluidic device configured for sampling, metering and recovering a metered amount of bodily fluid for analysis by capillary transport, the device comprising: an inlet section for receiving a bodily fluid sample, the inlet section including an inlet port arranged to receive a supply of bodily fluid; a metering function configured to receive the bodily fluid from the inlet section and including a first channel; and a subsequent section configured to receive the bodily fluid from the metering function and including a second channel, the first channel including a capillary stop valve configured to interrupt or reduce the flow of bodily fluid therethrough and visual inspection means arranged adjacent to the capillary stop valve, the shape and / or dimensions of the inlet port being configured such that when the supply of bodily fluid to the inlet port is removed, the Laplace pressure of the bodily fluid meniscus at the inlet port is higher than a threshold pressure of the capillary stop valve.

[0112] The shape and / or dimensions of the inlet port can be designed to achieve a desired curvature of the meniscus of the bodily fluid that adheres to the inlet port when the supply of bodily fluid is removed. In one embodiment, the bodily fluid is blood from a finger prick that is applied to the inlet port. The curvature of the meniscus then determines the Laplace pressure caused by the surface tension of the liquid. By selecting the shape and / or dimensions of the inlet port such that the Laplace pressure of the bodily fluid at the inlet port is higher than the threshold pressure of the capillary stop valve, the capillary stop valve will break when the supply of bodily fluid (e.g. a blood drop from a finger) is removed to allow the bodily fluid to flow from the first channel to the second channel. This can be used to measure the amount of bodily fluid before it flows into the second channel. The user can check the fill level with a visual inspection means to ensure that a sufficient amount has been delivered.

[0113] In one embodiment, the capillary stop valve is selected from at least one of a portion of the first channel having an altered hydrophilicity and / or a portion of the first channel having an altered dimension. The hydrophilicity and / or dimension of the first channel may be configured to achieve a desired threshold or breakthrough pressure of the capillary stop valve. Preferably, the capillary stop valve is formed by an abrupt increase in the height of the first channel.

[0114] In one embodiment, the subsequent section consists of at least one porous medium for receiving or withdrawing the bodily fluid from the first channel, so that a sample of the bodily fluid can be collected in a simple and efficient manner.

[0115] In one embodiment, the ratio of the height of the first channel to the second channel is at least 1.1: 1, preferably at least 2: 1. The height difference ensures continuity of capillary transport from the first channel to the second.

[0116] In one embodiment, the surface surrounding the inlet port is hydrophobic, which aids in the formation of droplets of bodily fluid that adhere to the inlet port, thereby increasing the Laplace pressure.

[0117] In one embodiment, the metering function is a blood pre-metering function, and the first channel is a pre-metering channel disposed in fluid communication with the filtration membrane and an extraction chamber configured to receive fluid from the filtration membrane and transport and fill the plasma metering channel. With the filtration membrane, extraction chamber and plasma metering channel, the device is further configured to autonomously separate, meter and collect plasma from the blood, preferably in a capillary means disposed in fluid communication with the plasma metering channel.

[0118] In one embodiment, the device further comprises a pinch-off means configured to separate the metered amount of bodily fluid, the pinch-off means comprising at least one vent hole located in the portion of the extraction chamber having the maximum height, the vent hole providing an effective separation of the metered amount from the remaining amount of bodily fluid.

[0119] In one embodiment, the pinch-off means comprises a pinch-off region in fluid communication with the at least one vent hole, located adjacent to the portion of the extraction chamber having the maximum height and surrounded by a region having a lower height. Preferably, at least one region surrounding the pinch-off region has a height lower than the height of the plasma metering channel. The surrounding region of reduced height leads to a reduction in the capillary pressure of the pinch-off region, thus facilitating the introduction of air bubbles.

[0120] In one embodiment, the device further includes a fluid connector extending between the extraction chamber and the plasma metering channel, and an air vent. The air vent may be located adjacent to where the fluid connector abuts the plasma metering channel, or may be located where the fluid connector abuts the plasma metering channel. Preferably, the air vent is located at the entrance to the plasma metering channel and configured as an orifice to ambient air having a cross-sectional area equal to or greater than the size of the cross-sectional area of ​​the plasma metering channel. Thus, the air vent is located in a location of the device with low capillary pressure that is optimal for introducing air bubbles downstream of the extraction chamber and upstream of the plasma metering channel to separate a metered amount of bodily fluid.

[0121] In one embodiment, the fluid connector has a different dimension than the plasma metering channel, the dimension being selected from one or more of height, width and length.

[0122] In one embodiment, the maximum height of the extraction chamber is less than the height of the plasma metering channel.

[0123] In one embodiment, the extraction chamber is approximately wedge-shaped with a gradually increasing height, the top of the extraction chamber being defined by the flat lower surface of the filtration membrane, and the hydrophilic floor of the extraction chamber extending from the contact with the filtration membrane toward the plasma metering channel at an acute angle. The acute angle between the filtration membrane and the floor of the extraction chamber allows for a wedge-shaped extraction chamber that diverges toward the plasma metering channel, thereby allowing the space between the diverging surfaces to be gradually filled, essentially forming a capillary pump. At the same time, the approximately flat and horizontal orientation of the filtration membrane can be maintained, facilitating the integration of the filtration membrane into the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction.

[0124] Further, a sixth aspect of the present disclosure relates to a method for sampling, transporting and retrieving a metered amount of a bodily fluid for analysis by capillary transport in a microfluidic device, the method comprising the steps of: manually applying the bodily fluid to an inlet port of the device; filling with the bodily fluid by capillary pressure a first channel disposed in fluid communication with the inlet port, the first channel comprising a capillary stop valve configured to interrupt or reduce the flow of the bodily fluid therethrough; and detecting when the first channel is properly filled. the capillary stop valve is configured to permit flow of bodily fluid therethrough; and receiving a metered amount of bodily fluid to be transported into a porous medium disposed in fluid communication with the first channel.

[0125] In one embodiment, the capillary stop valve is selected from at least one of: a portion of the first channel that has had its hydrophilicity altered; a portion of the first channel that has had its dimensions altered.

[0126] In one embodiment, the method further comprises collecting a metered amount of the bodily fluid in the porous medium acting as a capillary means.

[0127] The method facilitates sampling of bodily fluids by allowing the user to provide a sufficient amount of bodily fluid before the fluid is allowed to continue flowing through the device to be collected in the porous media.

[0128] In a seventh aspect of the present disclosure, a microfluidic device is provided that is configured to sample, meter, and collect a metered amount of bodily fluid for analysis by capillary transport with a means for separating the metered amount from the remaining bodily fluid across a filtration membrane for removing cells such as red blood cells. The device comprises an inlet section including an inlet port for receiving a sample of bodily fluid, the inlet section configured to transport the sample to the filtration membrane. The device further comprises a metering section including an extraction chamber arranged to receive the extracted bodily fluid from the membrane and a metering channel. The device also comprises an outlet section configured to receive, transport, and collect the filtered bodily fluid from the metering channel by capillary means. The metering section further comprises pinch-off means configured to separate the metered amount of filtered bodily fluid in the metering channel from the remaining bodily fluid in the extraction chamber, the pinch-off means consisting of at least one vent hole arranged in a portion of the extraction chamber having a maximum height. The vent hole provides an effective separation of the metered amount of bodily fluid from the remaining bodily fluid amount.

[0129] In one embodiment, the pinch-off means comprises a pinch-off region located adjacent the inlet of the metering channel and in fluid communication with the at least one vent, the pinch-off region comprising a height reduction element having a height less than the maximum height of the extraction chamber. Preferably, the extraction chamber comprises a gradually increasing portion, a portion having a height reduction element, and a portion of the extraction chamber in fluid communication with the metering channel where the maximum height of the extraction chamber is present. The height reduction element ensures that the pinch-off region has a height greater than adjacent portions of the extraction chamber, thereby reducing the capillary pressure in the pinch-off region and facilitating the introduction of gas bubbles.

[0130] In one embodiment, the extraction chamber is approximately wedge-shaped, with the top of the extraction chamber defined by the flat lower surface of the filtration membrane, and the hydrophilic floor of the extraction chamber extending from the contact with the filtration membrane toward the metering channel at an acute angle. The acute angle between the filtration membrane and the floor of the extraction chamber allows the wedge-shaped extraction chamber to be realized, which diverges toward the metering channel, thereby allowing the space between the diverging surfaces to be gradually filled, essentially forming a capillary pump. At the same time, the approximately flat and horizontal orientation of the filtration membrane can be maintained, which facilitates the integration of the filtration membrane in the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction. Preferably, the maximum height of the plasma extraction chamber exceeds the height of the metering channel.

[0131] In one embodiment, at least a portion of the extraction chamber surrounding the pinch-off region has a height that is less than the height of the metering channel. The surrounding region of lesser height provides a reduction in capillary pressure in the pinch-off region, thus facilitating the introduction of gas bubbles.

[0132] In one embodiment, the device includes through holes in the height reduction element to prevent liquid pinning in the extraction chamber.

[0133] In one embodiment, the metering section includes a brewing chamber having a portion of gradually increasing height, a portion having a height reducing element, and a portion having a maximum brewing chamber height disposed in fluid communication with the metering channel.

[0134] In one embodiment, the device comprises an inlet section including an inlet port and a channel system, and a filtration membrane configured to separate plasma from blood, the inlet section and channel system configured to transport a sample of bodily fluid to and distribute across the filtration membrane with stepwise or gradually increasing capillary action from the inlet section to the filtration membrane, with features as outlined in the preceding aspects of the disclosure, such as the second aspect.

[0135] In one embodiment, the device comprises a metering channel having an outlet section having a dimensional change configured to assume a shape that approximately matches the surface shape of a capillary means having the characteristics as outlined in the preceding aspects of the present disclosure, such as the third aspect, when a fluid front meniscus of a metered amount of separated bodily fluid is transported to the outlet section.

[0136] In one embodiment, the device comprises an outlet section having a matching hydrophilic porous bridge element insertable into the bridge cavity such that the bridge cavity is substantially filled with the features outlined in the preceding aspects of the present disclosure, such as the fourth aspect.

[0137] In one embodiment, the device is a multi-layer device having a wedge-shaped extraction chamber, the floor of which is continuous with the floor of the metering channel and extends at an acute angle from the underside of the filtration membrane, and the floor of the extraction chamber is inclined relative to the floor of the metering channel to form a gradient. The device may be manufactured using multi-layer arrangements and methods having the features as outlined in the preceding aspects of the disclosure, such as the fifth aspect.

[0138] In one embodiment, the device comprises an inlet having a pre-metering feature including a visual inspection means, and a capillary stop valve having features as outlined in the previous aspects of the disclosure, such as the sixth aspect.

[0139] In an eighth aspect of the present disclosure, a microfluidic device is provided that is configured to sample, meter, and recover a metered volume of bodily fluid for analysis by capillary transport with means for separating the metered volume from the remaining volume of bodily fluid across a filtration membrane for removing cells such as red blood cells. The device comprises an inlet section including an inlet port for receiving a sample of bodily fluid, the inlet section being configured to transport the sample to the filtration membrane. The device further comprises a metering section including an extraction chamber arranged to receive the bodily fluid extracted from the membrane, a metering channel, and a fluid connector arranged between the extraction chamber and the metering channel, and a pinch-off means including at least one vent configured to introduce at least one air bubble to separate the metered volume. The vent achieves effective separation of the metered volume from the remaining volume of bodily fluid.

[0140] In one embodiment, the extraction chamber has a gradually increasing height up to a maximum value that is less than the height of the metering channel.

[0141] In one embodiment, the fluid connector has a different dimension than the metering channel, preferably such dimension is selected from one or more of height, width and / or length.

[0142] In one embodiment, the fluid connector has a height that gradually increases to the maximum height of the metering channel. In a particular embodiment of the fluid connector, the fluid connector is disposed at a height lower than the maximum height at the inlet from the extraction chamber and the height gradually increases to the height of the metering channel.

[0143] In one embodiment, the device has at least one vent hole located in the metering section where the height exceeds the maximum height of the extraction chamber. In one embodiment, the at least one vent hole is located adjacent to or at the location where the fluid connector abuts the metering channel. In another embodiment, the at least one vent hole is located at the location where the height is maximum.

[0144] In one embodiment, the at least one vent is located at the entrance to the metering channel and is configured to have an orifice to ambient air having a cross-sectional area at least as large as the cross-sectional area of ​​the metering channel.

[0145] In one embodiment, the fluid connector joins the metering channel at a sharp angle or curve.

[0146] In one embodiment, the extraction chamber is approximately wedge-shaped, with the top of the extraction chamber defined by the flat lower surface of the filtration membrane, and the hydrophilic floor of the extraction chamber extending from the contact with the filtration membrane toward the metering channel at an acute angle. The acute angle between the filtration membrane and the floor of the extraction chamber allows for a wedge-shaped extraction chamber that diverges toward the metering channel, thereby allowing the space between the diverging surfaces to be gradually filled, essentially forming a capillary pump. At the same time, the approximately flat and horizontal orientation of the filtration membrane can be maintained, which facilitates the integration of the filtration membrane in the chamber structure to protect the blood sample from evaporation and contamination during plasma extraction. Preferably, the maximum height of the plasma extraction chamber exceeds the height of the metering channel.

[0147] Preferably, the extraction chamber, the fluid connector and the metering channel have the same hydrophilic floor.

[0148] In one embodiment, the device comprises an inlet section including an inlet port and a channel system, and a filtration membrane configured to separate plasma from blood, the inlet section and the channel system configured to transport a sample of bodily fluid to and distribute across the filtration membrane with stepwise or gradually increasing capillary action from the inlet section to the filtration membrane, with features as outlined in the preceding aspects of the disclosure, such as the second aspect.

[0149] In one embodiment, the device comprises a metering channel having an outlet configured with a dimensional change such that a fluid front meniscus of a metered amount of separated bodily fluid, when transported to the outlet section, assumes a shape that approximately matches the surface shape of a capillary means having the characteristics as outlined in the preceding aspects of the present disclosure, such as the third aspect.

[0150] In one embodiment, the device comprises an outlet section having a matching hydrophilic porous bridge element insertable into the bridge cavity such that the bridge cavity is substantially filled with the features outlined in the preceding aspects of the present disclosure, such as the fourth aspect.

[0151] In one embodiment, the device is a multi-layer device having a wedge-shaped extraction chamber, the floor of which is continuous with the floor of the metering channel and extends from the underside of the filtration membrane at an acute angle, and the floor of the extraction chamber is inclined relative to the floor of the metering channel to form a gradient. The device may be manufactured using multi-layer arrangements and methods having the features as outlined in the preceding aspects of this disclosure, such as the fifth aspect.

[0152] In one embodiment, the device comprises an inlet having a pre-metering feature including a visual inspection means, and a capillary stop valve having features as outlined in the previous aspects of the disclosure, such as the sixth aspect. [Brief description of the drawings]

[0153] The present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] A general overview of a microfluidic device adapted to extract plasma from whole blood by finger prick, transport and separate the blood, and extract a defined amount of plasma from the blood is shown. [Diagram 2] AH: Plasma sampling at several successive fluid processing steps is shown. [Diagram 3] A-D: A capillary force-driven microfluidic device with volumetric control of the applied sample fluid. [Figure 4]A-E: A capillary force-driven microfluidic device with a microfluidic feature introduced between the indicator window and the connecting capillary section for volumetric control of the applied sample fluid. [Diagram 5] A-G: Cross-sectional schematics of a microfluidic device using a capillary stop valve fabricated by layer-by-layer technology. [Figure 6] A-D: Capillary pressure balance in a microfluidic device according to an embodiment of the present disclosure. [Figure 7] AG: Cross-sectional views of a microfluidic device according to one embodiment of the present disclosure showing the different layers that form the pinch-off region. [Figure 8] 1A-C: Plan and cross-sectional views of a microfluidic device showing a pinch-off solution according to one embodiment of the present disclosure. [Figure 9] 1A-B are cross-sectional views of a microfluidic device showing a pinch-off solution according to one embodiment of the present disclosure. [Figure 10] 1A-B are cross-sectional views of a microfluidic device showing a pinch-off solution according to one embodiment of the present disclosure. [Figure 11] 1A-C: Plan and cross-sectional views of a microfluidic device showing a pinch-off solution according to one embodiment of the present disclosure. [Figure 12] FIG. 13 is a plan view of one embodiment of a microfluidic device that overcomes the problem of metering accuracy by using a fluid connector with a vent between the extraction chamber and the metering channel. [Figure 13] AD: Plan views of a microfluidic device including a fluidic connector and four different vent designs. [Figure 14] 5A-5F are cross-sectional views illustrating steps in a method for manufacturing a microfluidic device according to one embodiment of the present disclosure. [Figure 15] A-F: Generally, an embodiment of a microfluidic device having a channel system with stepwise increasing capillarity that allows determining when a sufficient volume of body fluid has been introduced. [Figure 16]AF: Shown are cross-sectional views of an embodiment of the present disclosure having a capillary stop valve disposed in fluid communication with a pre-metering channel. [Figure 17] 1A-B are cross-sectional views illustrating one embodiment of a method for manufacturing the outlet portion of a microfluidic device. [Figure 18] FIG. 13 is a top view showing an example of bubble formation near an outlet in a microfluidic device. [Figure 19] FIG. 13 is a top view showing successful transfer of liquid from a channel to a capillary means according to one embodiment of the present disclosure. [Figure 20] FIG. 2 is a cross-sectional view of a metering channel of a microfluidic device according to one embodiment of the present disclosure. [Figure 21] AB: Test results for narrowing metering channels in a microfluidic device according to one embodiment of the present disclosure. [Figure 22] 11A-C show test results for narrowed metering channels in a microfluidic device according to another embodiment of the present disclosure. [Figure 23] 11A-C show test results for narrowed metering channels in a microfluidic device according to another embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0154] The following sections provide a detailed description of a microfluidic device configured to sample and retrieve metered volumes of bodily fluids for analysis by capillary transport, in accordance with an embodiment of the present disclosure. In the drawings, like reference numerals indicate the same or corresponding elements throughout the several views. It will be understood that these figures are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0155] Example 1: Microfluidic Device 1 shows an exemplary embodiment of a microfluidic device adapted to extract plasma from whole blood by finger prick, transport and separate the blood, and extract a defined amount of plasma from the blood. In broad overview, the system comprises the following components arranged in the flow direction through the system as shown in FIG. 1: Entrance section 24, ·Entrance 4, · Channel system 25, A first channel 6, also called the pre-metering application channel; The second channel 8, also called the middle channel, a third channel 10, also called the filtration channel; ·Filtration membrane 12, the inlet section 24 including -Weight Section 26, Extraction chamber 14, 16 vent / pinch-off structures; 18 plasma metering channels, the metering section 26 including Exit section 28, an outlet port 21 (with a bridging capillary element 20); Capillary means 22, the outlet section 28 including Equipped with.

[0156] The plasma sampling proceeds through several successive fluid processing steps, which are described in Figures 2A-2H. In summary, the figures show the following: Figure 2A: A first, pre-metering application channel in the inlet section 24; 2B: Removal of the blood supply 30 after the front meniscus 36 of blood reaches the capillary stop valve 35 leads to the formation of a convex rear meniscus 32 of blood attached to the inlet port 4; FIG. 2C: Laplace pressure pushes the concave front meniscus 36 of blood over the capillary stop valve 35; FIG. 2D: Flow through the second intermediate channel 8 to the filtration membrane 12, simultaneously filling the filtration membrane, emptying the pre-metered application channel 6 and initiating plasma extraction; FIG. 2E: Filling the third, filtration channel, channel 10; FIG. 2F: Continuous filtration into the extraction chamber 14; FIG. 2G: Filling the plasma metering channel 18; and FIG. 2H: Absorption of a metered volume of plasma into the capillary means 22 with air bubble ingress at the vent / pinch-off structure 16.

[0157] As shown in Figure 2A, blood 30 is loaded into the premetered application channel 6 via the inlet port 4. When the premetered application channel 6 is completely filled, the blood supply to the inlet port is manually interrupted, thereby metering a defined amount (see Figure 2B). The intermediate channel 8 transports the blood from the premetered channel 6 towards the filtration channel 10 and filtration membrane 12 (see Figure 2C).

[0158] Therefore, the capillary pressure in the intermediate channel 8 needs to be higher than the capillary retention pressure that keeps the liquid at the inlet port, so that the liquid can be pumped from the premetered application channel 6 to the filtration channel 10 / filtration membrane 12. A high capillary pressure in the intermediate channel 8 is also beneficial to prevent air bubbles at the contact of the second channel with the filtration membrane 12, where a sudden increase in capillary pressure could introduce air bubbles into the intermediate channel 8. Air bubbles could disrupt the capillary action of the fluid plug moving through the system, thus stopping the fluid operation. Once the blood meniscus 32 contacts the filtration membrane / third channel 10, the filling of these two compartments occurs in parallel, following the capillary forces in either of the compartments (see Figures 2D-E).

[0159] Since the third channel 10 and the membrane 12 are arranged in parallel, the filtration membrane is usually filled first due to the higher capillary pressure in the filtration membrane. Once the void volume of the membrane is filled with blood / plasma, the third channel 10 starts / continues to fill. The filtration membrane 12 has a capillary gradient with pore sizes of a few tenths of a micrometer on the blood receiving side and 2-3 micrometers on the plasma extracting side. As soon as the plasma reaches the lower surface of the filtration membrane 12, extraction of the plasma into the extraction chamber 18 occurs due to the high capillary pressure at the intersection of the plasma filtration membrane 18 and the hydrophilic bottom substrate 38 (see FIG. 2D). The diverging (diverging) space between the membrane 12 and the hydrophilic bottom substrate 38 gradually fills with plasma because the capillary pressure in the extraction chamber 14 is much higher than the holding pressure in the premetered application channel 6 (see FIG. 2D-FIG. 2F).

[0160] Once the plasma meniscus reaches the entrance of the plasma metering channel 18, plasma continues to flow into the plasma metering channel 18 driven by the capillary pressure in the channel 18 (see FIG. 2G). The capillary pressure in the plasma metering channel 18 needs to be significantly greater than the retaining capillary pressure in the pre-metered application channel 6 to allow plasma filtration through the membrane 12. Once the plasma metering channel 18 is completely filled and the meniscus reaches the exit port 21, the sudden increase in capillary pressure draws the plasma through the exit port 21 and into the capillary means 22 (see FIG. 2H).

[0161] Due to the high flow resistance of blood in the filtration membrane, fluid absorption upstream of the filtration membrane is minimal. Instead, the provision of a vent / pinch-off structure 16 downstream of the filtration membrane results in a low bubble ingress resistance, leading to pinch-off and metering of the plasma volume. Since the presented system is based on a structure of a foil leading to the liquid-air interface of the downstream capillary system, the ingress of air bubbles is possible at some points. Therefore, it is important to take into account the capillary holding pressure in the downstream capillary system in order to perform a controlled and reproducible air bubble ingress that allows the volume of plasma to be metered with the desired accuracy. Plasma absorption through the outlet port continues until the entire plasma metering channel is emptied and the plasma volume is transferred to the capillary substrate.

[0162] Since there is no safety mechanism to prevent a second filling cycle of the plasma metering channel if there is too much blood on the filtration membrane, it is crucial to have a clearly defined input volume. The input volume is directly correlated with the dead volume of the system and with the plasma output volume of the system. For this purpose, instead of applying blood directly to the membrane, a pre-metering application channel 6 is introduced.

[0163] Another reason for introducing the pre-metered application channel 6 is that the total amount of blood required is about 70 μl. Since it is assumed that the user will not use a measuring device such as a pipette, but will instead inject blood by direct finger prick, the pre-metered application channel 6 allows for the collection of several successive drops of blood and gives the user feedback on the filling status of the device. Once enough blood has been injected into the system, the indicator area will show that filling was successful. The pre-metered application channel 6 is also nicely integrated with a third channel, whose purpose is to distribute the blood homogeneously across the membrane and limit the evaporation of water from the blood during filtration.

[0164] (Example 2) Pre-weighing A capillary force driven microfluidic device with flow control of injected sample fluid is generally depicted in Fig. 3A-3D. The device of Fig. 3A-3D is configured to collect one or more droplets at an inlet port 40 for transport to a first, pre-metered application, channel 42 having a pre-metered section / compartment. Once the pre-metered section is filled, a fill indicator 44 allows the user to confirm the fill status so that the supply of liquid to the inlet port 40 can be manually interrupted and a defined amount is trapped in the pre-metered compartment. The pre-metering operation is performed in four steps: (a) application of liquid to the inlet port 40, (b) capillary filling of the pre-metered compartment, (c) reaching and manually reading the indicator 44, and (d) removal of excess liquid from the inlet port 40.

[0165] Figures 3A-3D illustrate this process. Figure 3A shows liquid being applied to the inlet port 40. Figure 3B shows the capillary filling into the first channel or pre-metered compartment 42. Figure 3C shows the indicator 44 being reached and manually read. In Figure 3D, excess liquid is removed from the inlet port 40.

[0166] Manual interruption of the flow supply to the inlet is done with a fixed delay, thus introducing a defined amount of time-dependent overfill into the second channel or connecting capillary channel 46. The amount of overfill depends on the time between reaching the indicator window 44 and removing the liquid from the inlet port 42, and on the flow rate in the connecting capillary channel 46.

[0167] FIG. 4A shows the components of the capillary system, including the inlet port 50, the first channel 52 (also called the pre-metering channel), the indicator window 54, and the second channel 58 (also called the connecting or subsequent capillary channel). The introduction of other microfluidic features suitable for the capillary drive, such as a valve or flow reduction gate 56, can help to increase metering accuracy. Such microfluidic features can be introduced between the indicator window 54 and the second channel 58 to slow down or stop the flow between the two sections, as shown in FIG. 4B-E.

[0168] 4B-4E show the metering of liquid in a capillary system using a flow reduction gate or stop valve 56. The flow reduction gate acts to reduce the rate of flow significantly so that in a given time (e.g., 3 seconds) a smaller volume 57 spills out of the pre-meter channel 53 into the second channel 58 than would occur without the flow reduction gate, such that the amount of liquid applied to the capillary system is approximately equal to the metered amount 55 of liquid in the pre-meter channel 52. For example, a flow reduction gate can be implemented by changing the hydrophilic / hydrophobic properties of the microchannel, adjusting the dimensions of the microchannel, or changing the flow resistance of the microchannel.

[0169] Stop valves, such as dissolvable membrane valves or capillary stop valves, completely stop flow so that overfilling can be minimized. Dissolvable membrane valves break when exposed to liquid, allowing flow to be stopped for a period of time before opening fluid communication to downstream connecting capillary means. Capillary stop valves act as pressure barriers and can be used to completely block flow in a capillary system until wetting of the valve occurs or additional liquid pressure pushes the liquid past the pressure barrier. Such liquid pressure can be introduced in a variety of ways, for example, by applying hydrostatic pressure or by changing the conditions of the inlet port, e.g., changing the Laplace pressure / capillary pressure force at the inlet.

[0170] Manual removal of excess liquid from the inlet port can be used to introduce a change in Laplace pressure that leads to the breakage of the stop valve initiating flow into the second channel. The dimensions and surface properties of the entire capillary system are selected to allow the transport of liquid from the metering section to the connecting capillary section. The capillary stop valve does not actually close, but creates a pressure barrier in the capillary flow that breaks when a certain pressure is applied to the liquid. The valve does not physically close, but only closes by blocking the capillary flow, so we say the valve breaks rather than opens. For a capillary stop valve, the breakage pressure is a function of the surface energy of the liquid-gas-interface, the wettability by the fluid, and the geometric dimensions of the valve. It can therefore be predefined by appropriate design of the microfluidic structure.

[0171] As a result, the shape and / or dimensions of the inlet port can be configured such that when the supply of body fluid to the inlet port is removed, the Laplace pressure of the body fluid meniscus at the inlet port is greater than the threshold pressure of the capillary stop valve.

[0172] (Example 3) Sample volume control using a capillary stop valve 5A-5G show one embodiment of a microfluidic device using a capillary stop valve 64 for sample volume control as generally described in Example 2. 5A-5G show cross-sectional schematics of a microfluidic device using a capillary stop valve made by layering techniques. The device is constructed using structured layers that are layered together. In FIG. 5A, the inlet port 60, the metering channel 62, the capillary stop valve 64, the location of the indicator window 66, and the second channel 68 are shown in cross-section. When a droplet contacts the inlet port 60, the liquid is drawn into the metering channel 62 of the device until it reaches the capillary stop valve 64 (FIGS. 5B-5D). Separating the excess liquid from the volume of liquid in the metering channel 62 results in a small amount of liquid adhering to the inlet port 60 outside of the metering channel 62.

[0173] This volume curvature pushes the liquid in metering channel 62 beyond capillary stop valve 64, as shown by the arrow, because the Laplace pressure due to surface tension on the liquid is higher than the threshold pressure of capillary stop valve 64. The liquid then continues to flow into second channel 68 (FIGS. 5E-5F) because the capillary pressure at the front of the liquid flow direction is higher than the capillary retention pressure at the inlet port.

[0174] Example 4: Capillary pressure balancing in a microfluidic device 6A-6D generally illustrate capillary pressure balance in a microfluidic device according to the present disclosure. The microfluidic device autonomously filters the plasma fraction from whole blood by allowing absorption of whole blood into an inlet section 72, shown as compartment A, and then pumping / transporting the blood through a filtration element (membrane) 74 to a metering section (including an extraction chamber and a metering channel) and an outlet section (including a capillary means / pump) 76, generally shown as compartment B in FIG. 6A. All fluid transport in the device is based on capillary pressure. The condition for successful filtration of plasma is that the capillary pressure in compartment B 76 is greater than the retention pressure in compartment A 72, such that fluid transport from compartment A to compartment B occurs in light of all frictional forces of the system.

[0175] More specifically, embodiments of the present disclosure include a plurality of microfluidic devices as described above. Fluid is pumped through the system forming fluid plugs or columns that are pumped through the system using capillary pressure from the inlet to the outlet. To ensure that the fluid plugs flow continuously through the system, a pressure difference must always be applied between the capillary pressure of the liquid front flowing towards the outlet and the capillary pressure of the liquid end that drags the fluid plug (holding pressure). The capillary pressure at the meniscus where the system is filled changes throughout the filling operation and is defined by the contact angle of the interface, the surface tension of the liquid and the (smallest) channel / feature dimensions. The capillary holding pressure at the receding end is defined by the same parameters with the difference that the receding contact angle defines the curvature of the liquid-air interface and thus the capillary holding pressure. When the microfluidic device is composed of stacked layers, the capillary height is typically much smaller than the channel width and mainly defines the capillary pressure in the different sections. During the liquid flow into the first channel, the liquid is not trapped in a capillary but is rather freely available in the form of a droplet or any shaped liquid reservoir, which allows filling the first channel described above, which has the largest capillary height in the system and therefore, relatively speaking, causes the lowest capillary pressure.

[0176] Once blood inflow has ceased, an open air-liquid interface is formed that tracks the fluid plug and opposes the capillary pressure at the liquid front throughout the filling and filtering operations. To allow continuous capillary flow of the plug through the device, all compartments / channels following the liquid front need to exert a capillary pressure significantly greater than that at the trailing end.

[0177] (Example 5) Change in Capillary Height Example 5 is a detailed embodiment of the microfluidic device generally described in Example 4. The microfluidic device of Example 5 is made from a stack of structured foils with stepwise introduced capillary height changes, except for the wedge-shaped gradient. Stepwise decreasing the capillary height allows the fluid to fill without pinning at the step. However, stepwise increasing the capillary height causes pinning, which stops the formation of the capillary, which should be prevented to ensure continuous operation of the device. These design requirements result in stepwise decreasing of the capillary height throughout the system, except for the plasma extraction chamber, where successive increases in capillary height allow for gradual filling of the wedge structure before stepwise decreasing of the capillary height again. An example of the operation of the system can be seen in Figures 2A-2H, and the relevant capillary dimensions are listed in Table 1.

[0178] [Table 1]

[0179] Examples 6A and 6B below refer to embodiments of a microfluidic device having different solutions for pinching off a metered volume of bodily fluid in order to transport the correct metered amount for collection by capillary means at the outlet of the device.

[0180] (Example 6A) Metering 1: Pinch-off below the membrane The present embodiment of the disclosure relates to a pinch-off structure in a capillary system that allows the use of capillary forces to separate a fluid plug into two fluid plugs, preventing fluid communication between the two plugs, and more specifically allows the separation of a well-defined volume of plasma from a fluid plug consisting of whole blood and plasma.

[0181] To pinch off / separate liquid in a capillary driven system, it is necessary to introduce air bubbles into the system. Air bubbles can be introduced into the system at existing liquid-air interfaces, such as vents or other open sections. The wedge structure in the plasma extraction chamber is designed such that the side of the edge cannot be sealed due to fabrication constraints. However, to enable accurate metering of plasma, it is necessary to control the absorption of plasma and the ingress of air bubbles below the wedge. Due to the structure of the microfluidic device, the part of the wedge structure with the highest capillary height in the plasma extraction system is located downstream of the plasma separation membrane, which is suitable for the ingress of air bubbles into the system. In this embodiment of the present disclosure, a pinch-off structure is designed to take advantage of the relatively low capillary retention pressure in the plasma extraction chamber and control the exact location where air bubbles can enter the capillary system when the plasma contacts the capillary pump.

[0182] Both Figures 7A-7G and 9A-9B show pinch-off below the membrane. Pinch-off occurs once the plasma front reaches the capillary means, initiating immediate absorption of plasma from the capillary system. Because filtration of plasma through the filter occurs much slower than absorption of plasma from the system, absorption results in an air bubble growing at the point of lowest capillary pressure, which in both cases occurs in the section below the filtration membrane. This causes a "necking" at the highest part of the capillary height until the fluid plug extending between the plasma third channel and the plasma metering channel collapses and an air bubble starts to grow in the plasma metering channel. Creating a necking and pinch-off below the membrane is advantageous because there is no liquid-solid interface on the left and right sides of the necking region, preventing corner flows that could otherwise lead to a capillary connection between the two fluid plugs. The corners of the square microchannel have high capillary pressures, which can trap fluid there and leave a connection between the two fluid plugs. Another advantage of pinching off below the plasma filtration membrane is that the plasma must fill the pinch-off area a second time before it can refill the plasma metering channel. Relatively speaking, the capillary height here is at its highest level and therefore the capillary pressure is relatively low, so refilling occurs fairly slowly.

[0183] In the pinch-off of plasma below the membrane, narrowing the connection between the plasma extraction chamber and the plasma metering channel reduces the volume contained in the section designed for pinch-off, which can result in undesired absorption of plasma from the section to the left of the pinch-off area.

[0184] Absorption of plasma through the outlet port 21 of the system can occur not only from the pinch-off area 84 adjacent to the entrance of the plasma metering channel 18, but also from different areas below the membrane. This undesired absorption is reduced by the pinch-off structures 83, 84 shown in Figs. 7A-7G. The capillary height below the filtration membrane 81 is reduced by the height reduction element 83 in the area where absorption of plasma is undesired, clearly defining a pinch-off area 84 of surface area approximately 2 mm x 2 mm, with a maximum capillary height (in the plasma system) of 250 μm. To the right of the pinch-off area 84, the channel cover 80 reduces the capillary height to 150 μm, and to the left of the pinch-off area 84, the extension structure 83 of the channel cover 80 reduces the capillary height to less than 150 μm. In this way, unwanted absorption of plasma from the wedge-shaped extraction chamber 87 below the membrane 81 is prevented.

[0185] At the pinch-off of the plasma below membrane 81, plasma fills from extraction chamber 87 into plasma metering channel 18. After connection to porous plug 89 at outlet port 21, absorption of plasma in plasma metering channel 18 occurs through outlet port 21, forming a constriction between plasma extraction chamber 87 and plasma metering channel 18. The plasma constriction collapses between the third channel and the plasma metering channel, separating the two fluid volumes.

[0186] FIG. 7A shows a schematic longitudinal section of one embodiment of a microfluidic device having a pinch-off region 84 taken along line GG, while FIGS. 7B-7G show transverse cut lines AA, BB, CC, DD, EE, and FF, respectively. FIG. 7F shows the overlap between the bottom 82 of the plasma metering channel 18 and the ceiling 80 of the plasma metering channel 18, which defines the capillary height 88 of the plasma system. The pinch-off region 84 is defined by a reduction in the capillary height upstream (left in FIG. 7A) and downstream (right in FIG. 7A) of the pinch-off region 84. The pinch-off region has open sidewalls 86, forming a liquid-air interface beneficial to the ingress of air bubbles and preventing corner flow.

[0187] Pinch-off below the membrane according to the design shown in Figures 7A-7G occurs as follows.

[0188] Prior to wetting the porous plug 89 at the outlet 21, the pinch-off region 84 below the membrane 81 fills with plasma. Wetting of the porous plug 89 results in absorption of plasma from the pinch-off region 84, forming a constriction. Further absorption of plasma from the constriction region results in collapse of the constriction, separating the fluid in the plasma extraction chamber 87 from the fluid in the plasma metering channel 18. Air bubbles then enter the plasma metering channel 18 as fluid in the channel 18 is absorbed from the outlet port 21 of the device. Refilling of the pinch-off region occurs from the plasma extraction chamber 87 as plasma filtration continues.

[0189] Figure 9A shows a longitudinal cross section of an embodiment of a metering 1 solution in which the extraction chamber 102 is generally wedge-shaped with a horizontally disposed filtration membrane 100 as a ceiling and a gradient 104 formed by a hydrophilic floor 106 that extends at an acute angle from the contact with the filtration membrane towards the metering channel 108. Figure 9B is a transverse cross section taken along line AA and shows the filling of the plasma 109 in the pinch-off region prior to pinch-off due to the introduction of an air bubble.

[0190] (Example 6B) Metering 2: Use of a pinch-off structure inside the metering channel As an alternative to the metering 1 solution shown in Figures 9A-9B, in Figures 8A-8C and 10A-10B, the height H1 of the capillary below the membrane 98 can be made lower than the height H2 of the metering channel, which would prevent undesired absorption of plasma below the membrane, but instead favor the formation of air bubbles inside the metering channel 90 at the location of the vent 92. This is achieved by shifting the start of the slope 96 further outward from the membrane 98 to define a wedge-shaped extraction chamber formed between the hydrophilic channel floor 93 and the filtration membrane 98, as shown in Figures 8B and 10A. This allows the introduction of air bubbles by placing a vent structure 92 in the metering channel 90. This embodiment of the disclosure relates to the use of a pinch-off structure in the metering channel 90.

[0191] In Figure 10A, the maximum height H1 of the extraction chamber is less than the height H2 of the metering channel, and thus H2 is the height of the highest capillary in the metering channel 90. When pinch-off occurs, it causes an air bubble to be pulled in at the vent 92 adjacent the inlet of the metering channel 90 as the fluid in the metering channel 90 contacts the capillary means 94 at the outlet. Figure 9B is a cross-sectional view taken along line AA and shows the filling of the pinch-off region adjacent the vent 92 with plasma 109 prior to pinch-off due to the introduction of an air bubble.

[0192] 11A-11C show alternative embodiments of a microfluidic device having a pinch-off within the metering channel, where the metering channel is non-linear, e.g., approximately Z-shaped. FIG. 11A is a plan view of a microfluidic device in which a filtration membrane 110 is positioned above an extraction chamber, similar to the embodiment of FIG. 8A. An air vent 92 is positioned adjacent to the metering channel 90 where the metering channel 90 makes a 90 degree turn. This positioning increases the surface area of ​​the liquid-air interface at the air vent 92, as will be described in more detail below. FIGs. 11B and 11C are cross-sectional views taken along lines AA and BB, respectively, showing the structure of the microfluidic device.

[0193] Example 7: L-shaped metering channel Testing various prototypes revealed that in order to avoid the absorption of excess plasma from below the membrane, the pinch-off of the air bubble must be performed as early as possible, i.e. as close as possible to the junction of the extraction chamber and the metering channel. The unwanted absorption of plasma from below the membrane depends on the properties of the blood, i.e. the hematocrit, and is unacceptable. The unwanted absorption of plasma is the result of the resistance (or lack thereof) offered by the membrane compartment. This is caused by factors such as clogging of the membrane pores with red blood cells (RBCs) (thus depending on the hematocrit), interactions between the membranes, the channel bottom layer (slope) and the membrane.

[0194] Furthermore, this system functions well for blood with a hematocrit value of 55 or 45. However, when the hematocrit value is 35 or less, it has been observed that a portion of the plasma does not follow the desired flow path to the outlet, and the plasma metering becomes inaccurate. The lower the hematocrit, the fewer red blood cells clogging the membrane, resulting in lower membrane resistance. As a result, the plasma flows very quickly from the plasma extraction chamber into the metering channel, and it becomes difficult to pinch off the air bubbles.

[0195] By testing the prototype, it was found that one way to solve the metering accuracy problem is to use a fluid connector 124 between the extraction chamber 122 below the membrane 120 and the metering channel 128, as generally depicted in the embodiment of FIG. 12. The embodiment of FIG. 12 has a vent hole 126 that introduces air bubbles that pinch off as close as possible to the fluid connector 124 and enables the pinch-off to be performed as quickly as possible after introducing the air bubbles into the system. This reduces the excess HCT-dependent flow from the membrane compartment. It was also discovered that the shape of the vent hole in the L-shaped metering channel serves the role of how easily air bubbles can be introduced into the system. For air bubbles to be introduced into the vent hole, Fp < Fc, where Fp is the capillary force acting on the liquid at the vent hole 126 and Fc is the capillary force acting on the liquid at the outlet 129. If Fp > Fc, instead, the air bubbles are drawn out from the outlet 129. Therefore, it is desirable for Fp to be as small as possible. The factors contributing to Fp are the retention of the fluid at the edge of the vent hole 126, the capillary force, and the liquid-air interface of the vent hole with respect to others. It has been empirically demonstrated that the larger the liquid-air interface, the easier it is for air bubbles to enter. This is thought to be the result of the tendency of the liquid to contract to a minimum surface area due to surface tension.

[0196] 13A-13D show four different vent 126 designs, 13A has the smallest liquid-air interface 127a, 13B has a slightly larger liquid-air interface 127b that roughly corresponds to the dimensions of the metering channel 128, 13C has a larger angled liquid-air interface 127c, and finally 13D has the largest non-linear liquid-air interface 127d. In design A, the liquid must expand from a small liquid-air interface to a large liquid-air interface (cross-section of the metering channel). In B, the bubble is introduced from a liquid-air interface of the same cross-section during bubble formation. However, in C and D, the liquid-air interface at the vent is larger than the channel cross-section, so less force is required to introduce the bubble into the channel.

[0197] (Example 8) Manufacturing method One embodiment of the microfluidic device relates to enabling gradients in a microfluidic substrate to generate height gradients.

[0198] Initiating plasma flow through the plasma extraction membrane requires a force that can be passive (capillary driven) or actively exerted by applying an external force. One way to establish capillary flow is to place the plasma extraction membrane at an angle across the opening of a microchannel. The membrane forms an acute angle between the bottom and top of the channel, creating a capillary force-driven flow down the membrane, which is transported into the microchannel. The time it takes for a particular blood volume to pass through the membrane and extract plasma is typically in the range of several minutes and can vary, as it also depends on the hematocrit of the blood. Given this time, it is necessary to protect the blood sample from evaporation during extraction. From a usability perspective, it is also necessary to protect the blood volume from contamination. As a result, to enable products using microfiltration-based plasma filtration, the filtration membrane needs to be integrated into the chamber structure.

[0199] From a microfabrication point of view, it is difficult to incorporate uneven objects such as an obliquely oriented plasma membrane into a chamber structure because different height steps are created on the surface, making it difficult to seal the liquid.

[0200] Generally, plasma extraction membranes are constructed from soft polymeric materials or cotton fibers, so that the wedge structure does not provide a rigid support for subsequent layers to build upon. For incorporation into the chamber, it is preferable for the plasma extraction membrane to present a horizontal surface. To make this possible, it is necessary to create a slope on the microfluidic substrate in order to create a wedge structure between the channel and the membrane.

[0201] Common industrially scalable manufacturing techniques such as micro-injection molding, roll-to-roll hot embossing, and less scalable additive methods such as 3D printing, dispensing, and casting were considered. However, these methods were rejected as insufficient. First, it was difficult to find a supplier capable of manufacturing tools with gradients for injection molding or hot embossing, or casting. Second, none of these methods could produce the required sloped hydrophilic surface. For these methods, hydrophilic treatment is a prerequisite, which further complicates the manufacturing method. Finally, none of these methods were scalable. To overcome these challenges, a solution was developed to create the slope.

[0202] In particular, Example 7 shows a suitable method for creating height gradients in microfluidic channels in devices using foil substrates and layer-by-layer based fabrication techniques. By using thin foils, the foil substrate or a portion of it can be bent out of plane, allowing gradients that can be built into the microfluidic substrate.

[0203] A gradient can be created in the bottom substrate of a channel by isolating a portion of the microfluidic bottom substrate, attaching it to the bottom substrate as in A, and placing the other end of the isolated structure on a support structure as in B.

[0204] 14A-14F are cross-sectional views showing steps in a manufacturing method according to one embodiment of the present disclosure for manufacturing a plasma sampling system in the form of a microfluidic device. In order to incorporate a plasma extraction membrane into the chamber to prevent sample evaporation, protect against contamination and allow sample pre-metering, it is necessary to orient the plasma extraction horizontally instead of on an inclined plane, as shown in WO 2016 / 209147 A1, the contents of which are incorporated herein in their entirety. The proposed method of creating a gradient in the channel was implemented to form a wedge between the membrane and the bottom of the channel.

[0205] FIG. 14A shows a first layer in the form of a bottom substrate foil 130 with a first opening 131 for an extraction chamber extending between points a and b, and a second opening 133 at point c for accommodating a capillary means such as a paper substrate at the outlet.

[0206] 14B shows a second layer in the form of support structures 132 assembled onto the first layer forming a high plateau on the bottom substrate 130 adjacent point a of the first opening 131. The support structures 132 can be made from a dsPSA, a dispensed or screen printed polymer.

[0207] FIG. 14C shows a third layer in the form of a hydrophilic bed layer 134 assembled on the first and second layers. The third layer is intended to form a continuous floor of the extraction chamber and to integrally form a metering channel in fluid communication with the extraction chamber. For this purpose, the portion forming the floor of the extraction chamber is inclined with respect to the floor of the metering channel so as to form a slope 135. The free end of the slope 135 is supported and attached to the support structure 132 adjacent point a, while the remaining part of the bed layer 134 is attached to the base substrate 130 adjacent point b and extends towards and at least partially covers the second opening 133 adjacent point c. The slope thus extends across the first opening 131 between points a and b. The bed layer 134 may have an opening that aligns with the second opening 133 of the base substrate 130 when the two are assembled, thereby forming an outlet port 142. The third layer may be comprised of a hydrophilic foil material facing upward and an adhesive layer facing downward.

[0208] In one embodiment, the beveled surface 135 is formed by a slot in the floor layer 134 to define a tongue. The slot may be approximately C-shaped to define a substantially circular or substantially square tongue on three sides. In this case, as shown on the left side of FIG. 14C, the free end of the tongue is supported on the support structure 132 adjacent point a, while the portion of the floor layer 134 adjacent the free end of the tongue is attached to the bottom substrate 130.

[0209] 14D shows a fourth layer in the form of a channel structure layer 138 assembled on the third layer 134. The channel structure layer 138 includes openings to accommodate the support structures 132 and the angled ramps 135 that form the floors of the extraction chambers, and slots that form the sidewalls of the metering channels. The fourth layer may be cut from a double-sided PSA tape having the channel structures and membrane chamber openings.

[0210] 14E shows a fifth layer in the form of a channel cover layer 140 assembled onto the fourth layer. The channel cover layer 140 may be arranged to have an opening corresponding approximately to the size of the extraction chamber 137, with a portion of it attached to the support structure 132 adjacent the free end of the slope 135 of the bed layer 134. The fifth layer may be comprised of a hydrophilic surface facing downwards and a sticky surface facing upwards. The hydrophilic surface constitutes the top of the metering channel, and the sticky surface allows for additional layers to be attached on top of the channel cover layer 140.

[0211] 14F shows a five-layer structure that provides a flat top surface that facilitates subsequent assembly of filtration membrane 141 and additional structure 148 to form a chamber around filtration membrane 141. A slope 135 extending between points a and b forms a wedge-shaped extraction chamber 137 between bed layer 134 and plasma extraction / filtration membrane 141. Extraction chamber 137 reaches its maximum height at metering channel inlet 139 adjacent point b.

[0212] Further embodiments of the invention include the increased use and utilization of height gradients in microfluidic systems. Such further embodiments find use in the applications mentioned in the background. For example, gradient channels can be filled with either liquid or hydrogel to study diffusion effects.

[0213] 15A-15F show a generalized microfluidic device having an inlet port 152, a first pre-metered application channel 154, and a second intermediate channel 156. A droplet of bodily fluid 150 is applied to the inlet port and allowed to be transported by capillary action in the first channel 154. Once the fluid is transported to a visual inspection means 155, such as an indicator window, the fluid is observed by a user, who removes excess fluid from the inlet port 152, thereby allowing the fluid to be further transported to any porous medium, for example, for collection, analysis, or further processing. The device may further include a third filtration channel 158 having higher capillarity than the pre-metered application channel 154 and the intermediate channel 156. Here, the filtration channel 158 is placed in fluid communication with a porous plug 159, which may be, for example, a filtration membrane, or a lateral flow medium.

[0214] 16A-16F show a microfluidic device having a capillary stop valve 166 disposed in fluid communication with a metering channel 164. FIGS. 16A and 16B show how a droplet of bodily fluid 160 is applied to an inlet port 162 and transported by capillary action as a fluid stream within a first channel 163 (also called an application chamber) towards the metering channel 164. In FIG. 16C, the fluid front has reached the capillary stop valve 166, which can be inspected by a user by visual inspection means 168. In FIG. 16D, the user removes bodily fluid 160 from the inlet port 162, thereby forming a fluid column that establishes sufficient pressing force to overcome the capillary stop valve 166, allowing the fluid column to proceed further into a porous plug 167 (FIGS. 16D and 16E) and be collected in a capillary means 169 (FIG. 16F).

[0215] (Example 9) Manufacturing outlet part Disclosed herein is a method for connecting microfluidic channels to a paper substrate, which transports the liquid in the channel onto the paper, and which is amenable to mass production.

[0216] The method includes using a porous but highly compressible material that conforms to the shape of the drain hole and can be compressed to allow the paper substrate to contact the adhesive on the bottom of the channel substrate. The porous material is placed into or over the hole and then compressed. Materials that can be used for the porous plug include, for example, micro paper pulp, micro fibrillated cellulose (MFC), open cell hydrophilic polymer foam, or highly compressible glass fiber web.

[0217] 17A and 17B are cross-sectional views of one embodiment of a manufacturing method using a glass fiber web, before and after assembly. In FIG. 17A, the outlet of the microfluidic device is shown forming a cavity 172 at the tip of a plasma metering channel 170 that terminates in a drain hole 171. A porous plug 174 made of glass fiber material is placed adjacent to the drain hole 171 to form a bridge element between the metering channel 170 and a capillary means such as a paper substrate 176. The porous plug 174 is cut smaller than the paper substrate 176 to allow for bonding of the substrate 176 to an adhesive surface 178 on the underside of the floor layer of the microfluidic device, but larger than the drain hole 171 to prevent a gap between the porous plug 174 and the drain hole 171.

[0218] 17B, ​​the porous plug 174 is inserted into and substantially fills the cavity 172 by applying pressure to the porous plug 174 and paper substrate 176. To this end, the porous plug 174 is oriented to conform to the shape of the cavity 172. In one embodiment, the porous plug 174 is formed of a compressible material, which allows it to enter the exit hole 171 and then expand within the cavity 172. Compression of the glass fiber adjacent the exit hole 171 as a result of the applied pressure is shown in bold.

[0219] In another embodiment, a dispensable material is dispensed into the outlet hole 171 and then solidifies to form a porous plug 174. The volume of material adapts to achieve the same result, i.e. to reach a bridge element that conforms to the shape and nearly fills the cavity 172, while at the same time preventing the formation of voids in the outlet shape, and at the same time allowing adhesion between the paper substrate 176 and the bottom of the microfluidic device.

[0220] The special design of the system solves several difficult problems in transferring liquid from the channel to the paper, namely, the use of highly compressible or ejectable materials reduces the need for precision cutting and placement of porous plugs in the exit holes. As a result, it allows for applications in automated high-throughput manufacturing. In this example, glass fiber material and 6 mm paper disks were punched with diameters of 3 mm and 6 mm, respectively. The two disks were placed over the 2 mm diameter exit holes and simply aligned by eye. Furthermore, this solution eliminates the need for a PVA coating on the collection substrate, reducing the cost of the technology.

[0221] (Example 10) Straightening the Meniscus The various flow profiles of liquids in rectangular microchannels depend on the geometry of the channel and the interaction of the liquid with the channel material. The flow in the channels of the microfluidic devices of the present disclosure is shear-driven. Corner flow is affected by the corner angle and the wetting contact angle. To maintain continuous flow in the microchannels, the formation of air bubbles needs to be avoided.

[0222] Figure 18 shows an example of bubble formation using a porous plug at the outlet. The liquid meniscus impinges on the porous plug at its bottom, expanding the bubble at the top of the plug. In this embodiment of the disclosure, the porous plug is made of a glass fiber web.

[0223] Figure 18 shows the sequence of events when the meniscus of liquid in a channel encounters a porous plug inserted into the outlet hole of the channel. Due to the geometric mismatch between the meniscus and the porous plug, the initial impact occurs at the bottom of the plug, drawing air into the system and forming an air bubble that expands into the channel. Since the goal is to transport the liquid from the channel to the paper, the presence of the air bubble can block and interrupt the flow, and if the liquid in the channel to be emptied is being metered, the presence of the air bubble will result in a decrease in the amount metered.

[0224] By adapting the shape of the front meniscus of the fluid to the shape of the capillary means so that the shapes of the interfaces match each other, the formation of air bubbles can be avoided.

[0225] To prevent air bubbles from forming during the interaction between the porous plug and the liquid meniscus, the width of the metering channel is expected to be reduced. This reduction in width causes the liquid meniscus to change from a convex shape to a nearly straight, planar shape. At the same time, the curvature of the porous plug interface is also linearized by the reduction in the channel width. As a result, the shapes of the interfaces are consistent with each other.

[0226] 19, an example of successful use of the proposed invention to transport liquid from a channel 190 to a paper substrate 194 is shown. This example uses a 3 mm diameter glass fiber material as the porous plug 192 and a 6 mm diameter paper disk substrate 194. In a first region, the channel 190 has a width of approximately 2 mm, in a second region the width of the channel 190 gradually narrows, and in a third region the channel 190 has a width of approximately 1 mm.

[0227] The narrowing of the outlet allows the liquid meniscus to be reformed into a straight liquid front, facilitating control of the collision with the porous plug and preventing air bubble formation on collision between the two media. The solution using the glass fiber disk proved robust in further investigations and was successfully used for plasma extraction and quantification of whole blood in the hematocrit range of 30-55 HCT.

[0228] Furthermore, this solution can be easily adapted to other downstream systems for integration into point-of-care and rapid diagnostic testing systems.

[0229] A cross-section of the metering channel of the presently disclosed microfluidic device is shown in Figure 20. The top and bottom materials are composed of hydrophilic foil, and the sidewalls of the channel are composed of double-sided pressure-sensitive adhesive tape (dsPSA).

[0230] In this microfluidic device, the channel materials (bottom, top, sidewalls) that create the sidewall properties (roughness, wettability after cutting, corner angle) and the cutting method affect the meniscus shape, which is important to avoid entraining air bubbles when connecting with the glass fiber bundle at the outlet.

[0231] Various combinations of these parameters were tested to find the optimum combination to obtain a meniscus shape that matches the shape of the exit fiber bundle at the point where they join to obtain a bubble-free connection.

[0232] The following parameters: Both top and bottom are made of hydrophilic material (hydrophilicity level is A) <B<C) A.PCS B. Tesa C. Corbem Polyester Film ·Sidewall materials (various double-sided adhesive tapes) D. Tesa E. Our own products F.PCS G.AR Care H. AR Seal ·Cutting method I, Knife Plot J.Laser A K.Laser B Narrow exit hole L.1mm M.0.7mm N.0.4mm

[0233] (result) Figures 21A and 21B show tests using a channel that is 2 mm wide and gradually narrows to 1 mm in the area adjacent to the outlet. The material at the bottom and top of the channel is Coveme and the sidewalls are AR seal. A Laser A cutting method was used. Figure 21A shows a nearly planar meniscus in the 2 mm wide metering area, while Figure 21B shows a convex meniscus in the 1 mm wide area after narrowing.

[0234] Figures 21A and 21B show tests using a channel that is 2 mm wide and gradually narrows to 1 mm in the area adjacent to the outlet. The material at the bottom and top of the channel is from Coveme, and the sidewalls are a proprietary double-sided pressure sensitive adhesive tape. A knife plot cutting method was used. Figure 22A shows a concave meniscus in the metering channel in the 2 mm wide metering area, and in Figure 22B the meniscus is still concave after narrowing the channel width to 1 mm. In Figure 22C the meniscus has flattened after further reducing the channel width to 0.4 mm in the area adjacent to the outlet.

[0235] Figures 21A-21B and Figures 22A-22C show how different menisci can be produced by using the same hydrophilic foil cover in combination with two different cutting methods and materials. The meniscus obtained at the constriction in Figure 21B does not allow a bubble-free connection to the fiber bundle due to its convex nature, due to the inconsistent surface. A bubble-free connection does not appear even in the 2 mm area with a straight meniscus. In Figures 22A-22C, the width of the outlet constriction had to be reduced to 0.4 mm (Figure 22C) in order to flatten the plasma meniscus and make it conform to the fiber bundle surface. However, the width of the constriction was too small to allow effective contact and evacuation through the fiber bundle.

[0236] Figures 23A-23C show the implementation of this solution in the presently disclosed microfluidic device. Figures 23A-23C show tests using a channel that is 2 mm wide and gradually narrows to 0.7 mm in the area adjacent to the outlet. The material on the bottom and top of the channel is from Tesa, and the sidewalls are a self-made double-sided pressure-sensitive adhesive tape. A Laser B cutting method was used. In Figure 23A, a concave meniscus is formed in the metering channel and wobbles a bit as it advances through the metering channel. In Figure 23B, after reducing the channel width to 0.7 mm, the meniscus flattens and becomes less wobbly, while in Figure 23C, after further advancement, the meniscus straightens and conforms to the glass fiber bundle, allowing for bubble-free connection and evacuation.

[0237] Having described embodiments of a microfluidic device configured to sample, meter, and collect a metered volume of bodily fluid for analysis by capillary transport and corresponding methods according to the present disclosure, those skilled in the art will appreciate, however, that variations may be made within the scope of the appended claims without departing from the spirit of the invention.

[0238] All of the alternative embodiments or parts of the embodiments described above can be freely combined without departing from the inventive concept, so long as the combination is not inconsistent. [Explanation of symbols]

[0239] 2. Microfluidic Device 4 Inlet Port 6 First Channel (Pre-metered Dispensing Channel) 8 Second Channel (Middle Channel) 10 Third channel (filtration chamber) 12 Filtration membrane 14 Extraction chamber 16 Ventilation hole structure / pinch-off structure 18 Plasma Metering Channels 20 Porous Bridge Element 22 Capillary Means 24 Entrance Section 25 Channel System 26 Weighing Section 28 Exit Section 30 Body fluids (blood) 32 Fluid rear meniscus 35 Capillary Stop Valve 36 Fluid front meniscus 38 Hydrophilic bottom substrate 40 Inlet Port 42 First channel (pre-metered application channel) 44 Indicator window 46 Second channel (connecting the capillary channel) 50 Inlet Port 52 Pre-weighing Channel 54 Indicator 55 Measured quantity 56 Flow reduction gate (capillary stop valve) 57 Overflow amount 58 Second Channel (Subsequent Channel) 60 Inlet Port 62 First channel (pre-metered application channel) 64 Capillary Stop Valve 66 Indicator window 68 Second Channel (Subsequent Channel) 72 Section A 74 Filter element 76 Section B 80 Channel Cover 81 Filtration membrane 82 Hydrophilic floor 83 Height reduction element 84 Pinch-off structure 85 Incline 86 Opening side wall 88 Capillary Height 89 Porous Plug 90 metering channels 92 Air Vents 93 Hydrophilic channel floor 94 Porous Plug 95 Tilt 98 Filtration membrane 100 filtration membrane 102 Extraction chamber 104 Incline 106 Hydrophilic channel floor 108 metering channels 109 Plasma 110 Filtration membrane 120 Filtration membrane 122 Extraction chamber 124 Fluid Connector 126 Ventilation hole 127a Liquid-air interface 127b Liquid-air interface 127c Liquid-Air Interface 127d Liquid-air interface 128 metering channels 129 Exit 130 1st layer (bottom substrate foil) 131 First opening ab 132 Second layer (support structure) 133 Second opening c 134 Third layer (hydrophilic floor) 135 Slope (floor of brewing chamber) 136 Metering Channel Floor 137 Extraction Chamber 138 4th layer (channel structure) 139 Entrance to the metering channel 140 5th layer (channel cover) 141 Filtration membrane 142 Exit Port 148 Chamber Structure 150 Body Fluids 152 Inlet Port 154 First channel (pre-metered application channel) 155 Visual Inspection Methods 156 Second Channel (Middle Channel) 158 Third (filtration) channel 159 Porous Plug 160 Body Fluids 162 Inlet Port 163 First channel (pre-metered application channel) 164 Second Channel (Subsequent Channel) 166 Capillary Stop Valve 167 Porous Plug 168 Visual Inspection Methods 169 Capillary Means 170 metering channels 171 Exit hole 172 Cavity 174 Porous Plug 176 Paper Substrate 178 Adhesive surface 190 Channels 192 Porous Plug 194 Paper disc substrate

Claims

Claims 1. A microfluidic device (2) configured to sample a body fluid (30) and, by capillary transport, meter and collect a filtered and metered volume of the body fluid for analysis, comprising: an inlet section (24) for receiving a sample of the body fluid; a filtration membrane (12; 81) configured to separate plasma from blood; a metering section (26) configured to meter a predetermined amount of the filtered body fluid and isolate it from the remaining fluid within the device; and an outlet section (28) configured to receive and collect the metered volume of the body fluid from the metering section (26), the outlet section including capillary means (22) for collecting the metered volume of the body fluid; characterized in that the metering section (26) includes an extraction chamber (14) configured to receive the body fluid filtered from the filtration membrane (12; 81) and arranged to be in fluid communication with a metering channel (18); and the metering section (26) includes pinch-off means configured to isolate the metered volume of the body fluid, the pinch-off means including at least one air hole (16) disposed at a portion having the maximum height of the extraction chamber (14); the inlet section includes an inlet port (4) and a channel system (25) configured to transport a sample of the body fluid, the channel system (25) extending from the inlet port (4) to the filtration membrane (12; 81) and including a first channel (6), a second channel (8), and a third channel (10) arranged to be in fluid communication with the inlet port (4) in the flow direction in succession, the inlet section (24) and the channel system (25) being configured to transport a sample of the body fluid (30) to the filtration membrane in a capillary action that increases stepwise or progressively from the inlet section (24) to the filtration membrane (12; 81) and to distribute it throughout the filtration membrane (12; 81). Claims 2. The device according to claim 1, wherein the stepwise or progressive increase in capillary action of the channel system (25) is established by successively decreasing the height of the channels (6, 8, 10) continuously from the inlet port (4) to the filtration membrane (12; 81) and / or by successively increasing the hydrophilicity of the channels (6, 8, 10). Claims 3. ​ The device according to any one of claims 1 or 2, wherein the floor of the third channel (10) is defined by the flat upper surface of the filtration membrane (12; 81).

4. The device according to claim 2, wherein the ratio of the height of the first channel (6) to the second channel (8) is at least 1.1:1, preferably at least 2:1, and the ratio of the height of the second channel (8) to the third channel (10) is at least 1.1:1, preferably at least 2:

1. Preferably, the height of the first channel (6) is 500 to 2000 μm, the height of the second channel (8) is 100 to 600 μm, and the height of the third channel (10) is 25 to 200 μm.

5. The device according to claim 1, wherein the second channel (8) includes visual filling inspection means such as a capillary stop valve (35) and an inspection window (44), both of which are arranged adjacent to the first channel outlet.

6. The device according to claim 5, wherein the capillary stop valve (35) is selected from at least one of a part of the second channel (8) with changed hydrophilicity and / or a part of the second channel (8) with changed dimensions.

7. The device according to claim 6, wherein the capillary stop valve (35) includes a sharp increase in the height of the second channel.

8. The device according to claim 1, wherein the pinch-off means includes a pinch-off region (84) arranged to be in fluid communication with one or more air holes arranged in front of the inlet to the metering channel (18), and the pinch-off region (84) includes a height reduction element (83) having a height lower than the maximum height of the extraction chamber (14; 87).

9. The device according to claim 8, including a through hole in the height reduction element (83).

10. The device according to claim 9, wherein the extraction chamber (14; 87) includes a portion with a gradually increasing height, a portion having the height reduction element (83), and a portion having a maximum height, arranged to be in fluid communication with the metering channel (18).

11. In the apparatus according to claim 1, the roof of the extraction chamber (14; 87) is defined by the flat lower surface (85) of the filtration membrane (12; 81), and the floor of the extraction chamber extends at an acute angle from the contact portion with the filtration membrane (12; 81) to the metering channel (18). Apparatus.

12. In the apparatus according to claim 11, the extraction chamber (14; 87) is generally wedge-shaped with a gradually increasing height from the contact point with the filtration membrane (12; 81) towards the metering channel (18), and the maximum height of the extraction chamber (14; 87) exceeds the height of the metering channel (18). Apparatus.

13. In the apparatus according to claim 1, the first channel (6) has a dead volume and a volume correlated with the metered amount (output amount) of the apparatus. Preferably, the volume of the first channel (6) is sufficient to prevent the front meniscus (36) of the body fluid volume other than the metered amount from reaching the capillary means (22) of the outlet section (28). Apparatus.

14. In the apparatus according to claim 1, the metering channel (190) has an outlet with a dimensional change configured such that when transported to the outlet section (28), the fluid front meniscus of the separated metered amount of body fluid takes a shape substantially conforming to the surface shape of the capillary means (194). Apparatus.

15. In the apparatus according to claim 14, the dimensional change includes a decrease in the width and / or height of the metering channel (190). Apparatus.

16. In the apparatus according to claim 15, the distal end of the outlet of the metering channel (190) adjacent to the capillary means (194) has a constant width smaller than the width of the metering channel (190). Equipment.

17. In the apparatus according to claim 16, the outlet of the metering channel (190) has a first portion with a gradually decreasing width and a second portion with a constant width smaller than the width of the metering channel (190). Apparatus.

18. In the apparatus according to any one of claims 14 to 17, the surface shape of the capillary means (194) at the interface with the fluid front meniscus is a curved surface or substantially a plane. Apparatus.

19. In the apparatus according to claim 1, the outlet section comprises a hydrophilic porous bridge element (20; 167; 174; 194) having an average pore size smaller than the minimum dimension of the metering channel, and the bridge element is arranged in fluid communication with the outlet of the metering channel (18; 164; 170; 190) and the capillary means. Device.

20. A method for sampling, transporting, and collecting a filtered and metered volume of body fluid (30) for analysis by capillary transport within a microfluidic device (2), the following steps: Supplying a body fluid to an inlet port (4) of the device; Filling a channel system (25) arranged in fluid communication with the inlet port, the channel system comprising a first channel (6), a second channel (8), and a third channel (10) arranged in fluid communication with the inlet port in a flow direction. Continuously, the step of filling; Transporting, stepwise or progressively, a body fluid sample with increasing capillary action in the channel system from the inlet port to a filtration membrane (12; 81) configured to separate plasma from blood, spaced from the inlet port; Distributing the body fluid sample across the filtration membrane; Receiving the filtered body fluid into a metering section (26) including an extraction chamber (14) configured to receive the filtered body fluid from the filtration membrane and a metering channel (18) in fluid communication with the extraction chamber; Transporting the filtered body fluid within the metering channel to an outlet section (28) including capillary means (22) for recovering the filtered body fluid; Detaching the metered volume of body fluid by introducing at least one air bubble into a part of the metering section that induces a minimum capillary pressure; and Recovering the filtered and metered volume of body fluid within the capillary means; A method comprising.

21. A method according to claim 20, implemented using a blood sample for metering and collecting plasma using the apparatus (2) according to any one of claims 1 or 2.

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