Microchannel with electrode sensor

Abstract

Various embodiments of the present invention provide microchannel and electrode configurations that achieve uniform current density within the microchannel. The present invention provides this and other advantages.

Description

[Technical field]

[0001] The present invention relates to devices and methods for generating electrical current in a microchannel using uniplanar electrodes, where the current density is sufficiently uniform to provide sensitive detection and measurement of electrical properties within the microchannel. Thus, various embodiments of the present invention provide microchannel and electrode configurations that allow for sensitive measurement of changes in electrical properties (e.g., resistance, capacitance, inductance, impedance, dielectric constant) of and across a medium within a microchannel. The devices and methods described herein can be used in a variety of applications, including for assessing coagulation of blood samples. [Background technology]

[0002] The use of microfluidics for diagnostics and other applications is becoming increasingly common. The use of electrodes (e.g., electrode sensors) within microfluidic devices to measure changes in electrical properties as a readout is a useful approach because they tend to be robust and low cost.

[0003] There are many factors that affect the sensing ability of an electrode sensor in a microchannel: the shape, size, orientation, and material of the electrode, as well as the characteristics of the microchannel, are factors that affect the level and distribution of electrical current within the microchannel, and each factor in turn can affect the ability of the electrode to sense changes in the medium contained within the microchannel, whether that medium is a liquid, semi-solid, or air. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need in the art for microfluidic devices having electrodes that supply current within a microchannel, where the current is of sufficient density and sufficient uniform distribution such that the electrodes can sensitively detect changes in electrical properties within a medium within the microchannel, including changes that are small in magnitude and / or that may occur at different locations within the microchannel (e.g., including locations not proximate to the electrodes). Furthermore, there is a need in the art to provide such microchannels with single-planar electrodes, as single-planar electrodes are less complex to manufacture than multiplanar electrodes, and may enable a greater variety of (and potentially less expensive) manufacturing techniques. [Means for solving the problem]

[0005] An embodiment of the present invention provides a microchannel with a single-planar electrode. In a particular embodiment, the microchannel has a hexagonal shape with six edges and has two electrodes in a single-planar configuration, the two electrodes oriented to face each other on the same plane within the microchannel. In some embodiments, the two electrodes each have a rectangular portion oriented to be parallel to one of the six edges of the microchannel, the rectangular portion being oriented to be parallel to the rectangular portion of the other electrode. In further embodiments, one or more of the following features are present: the ratio of electrode height:channel height is at least 0.1; the ratio of electrode spacing:channel width is at least 0.5; each of the two electrodes has two circular regions, each circular region being located at an electrode end.

[0006] In certain embodiments, the ratio of electrode height:channel height for such microchannels is at least 0.1, at least 0.3, at least 0.5, at least 0.6, at least 0.8, or at least 0.9. In certain embodiments, the ratio of electrode height:channel height is in the range of 0.3 to 0.5. In some embodiments, the ratio of electrode height:channel height is 0.5 or greater.

[0007] In certain embodiments, the ratio of electrode spacing (the distance between the electrodes measured between the rectangular portions of the electrodes - see FIG. 6A(ii) (electrode spacing)) to channel width is 0.5 or greater. In further embodiments, the ratio of electrode spacing:channel width is 0.6 or greater, or 0.7 or greater. In certain embodiments, the ratio of electrode spacing:channel width is 0.8 or greater. In further embodiments, the ratio of electrode spacing:channel width is 0.9 or greater.

[0008] In a particular embodiment where each of the two electrodes has a circular region at each end, each circular region has a diameter ranging from 300 microns to 600 microns. In a further embodiment, each circular region has a diameter of 500 microns. Each electrode has an electrical via disposed within one of the circular regions.

[0009] In certain embodiments, the microchannel volume is 2.5 microliters or less. In further embodiments, the microchannel volume is 2.0 microliters or less.

[0010] In certain embodiments, the microchannels are fabricated using one or more minimally thrombogenic materials (e.g., polyimide, etc.) The microchannels may be fabricated such that the interior surfaces of the microchannels (and the surfaces that contact the sample) are minimally thrombogenic.

[0011] In certain embodiments, the electrodes comprise a thrombogenic material. For example, the electrodes may be made of a thrombogenic material and / or may include a coating, the coating comprising a thrombogenic material (such coatings may be referred to as thrombogenic coatings). Thrombogenic substances that may be used in the coating include, for example, metals such as gold, silver, platinum, etc. The use of such coatings makes the surface of the electrodes more susceptible to thrombosis.

[0012] An embodiment of the present invention also provides a hexagonal microchannel having two electrodes of the same shape and oriented opposite each other on the same plane within the microchannel. Each electrode may have a rectangular or straight portion parallel to one of the six edges of the microchannel and located within 500 microns of the parallel edge. Furthermore, in certain embodiments, the distance between the electrodes (the distance between the rectangular or straight portions of the electrodes) is at least 4,000 microns, and in further embodiments, the length of each edge is 4,000 microns or less. In certain embodiments, the ratio of electrode height:channel height of such a microchannel is at least 0.1, at least 0.3, at least 0.5, at least 0.6, at least 0.8, or at least 0.9, and in certain embodiments, the ratio of electrode height:channel height is 0.5 or more. In further embodiments, the microchannel can be made of a minimally thrombogenic material such that the inner surface of the microchannel (and the surface in contact with the sample) is minimally thrombogenic. In additional embodiments, the electrodes may be made of and / or include a coating, where the coating consists of a thrombogenic material. In certain embodiments, the microchannel volume is 2.5 microliters or less, and in further embodiments, the microchannel volume is 2.0 microliters or less.

[0013] The microchannel-electrode configurations provided herein provide uniform current distribution throughout the microchannel upon application of voltage. For example, in certain embodiments, when an excitation voltage is applied to a microchannel containing a medium having electrical properties (e.g., electrical conductivity) similar to that of non-clotted blood, the current density across at least 70% of the cross-sectional area of ​​the microchannel does not change by more than 20%. In further embodiments, the current density across at least 80% of the cross-sectional area of ​​the microchannel does not change by more than 20%. Furthermore, in certain embodiments, the current density across at least 80% of the microchannel volume does not change by more than 20%.

[0014] The present invention, as described herein, is directed to these and other important aspects. [Brief description of the drawings]

[0015] [Figure 1] Schematic diagram of current flowing through blood and how electrodes detect changes in electrical impedance. [Figure 2A-1] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2A-2] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2A-3] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2A-4] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2B-1] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2B-2] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2C-1]Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2C-2] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2C-3] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2D-1] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2D-2] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 2D-3] Various configurations of microchannels containing electrodes, with different electrode orientations and shapes and different microchannel geometries are provided and current generation within the simulated microchannels is shown. [Figure 3A-1] Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 3A-2] Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 3A-3] Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 3B-1]Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 3B-2] Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 3C] Two configurations of microchannels containing electrodes are provided, and the simulated current generation in each microchannel configuration is shown. [Figure 4A-1] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 4A-2] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 4A-3] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 4B-1] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 4B-2] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 4B-3] FIG. 1 is a schematic diagram showing simulated current generation and thrombus detection in two different configurations. [Figure 5A] 4 provides thrombus detection data for the six configurations described in Example 4. [Figure 5B] 4 provides thrombus detection data for the six configurations described in Example 4. [Figure 5C] 4 provides thrombus detection data for the six configurations described in Example 4. [Figure 5D] 4 provides thrombus detection data for the six configurations described in Example 4. [Figure 5E] 4 provides thrombus detection data for the six configurations described in Example 4. [Figure 5F]4 provides thrombus detection data for the six configurations described in Example 4. [Figure 6A-1] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6A-2] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6B-1] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6B-2] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6B-3] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6B-4] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. [Figure 6B-5] Simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration are provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 1A-1C are schematic diagrams of current flow through blood and how electrodes detect changes in electrical impedance. Electrical impedance can arise from ohmic resistance as well as capacitive and inductive reactance of components of the blood sample. For example, in FIG. 1A, blood contains red blood cells, Cm is the capacitance of the membrane of the red blood cells, Ri is the internal resistance of the red blood cells, and Rp is the resistance of the plasma. FIGS. 1B and 1C are schematic diagrams showing the flow of current through a blood sample containing red and white blood cells. FIG. 1B shows non-clotted blood, with cells separated. FIG. 1C shows clotted blood, with red and white blood cells clumping together. In both FIG. 1B and FIG. 1C, the solid arrows represent low frequency current and the dashed arrows represent high frequency current. These schematic diagrams show how low and high frequencies can be used to detect and measure changes in electrical impedance (or other electrical properties), for example, when a voltage is applied to the sample.

[0017] 2A-2D provide various configurations of microchannels containing electrodes with various electrode orientations and shapes and various microchannel geometries, and show current generation in the simulated microchannels. Simulations performed with various configurations show how the current distribution in the microchannel changes as a result of the microchannel geometry and the electrode geometry and placement. None of these configurations provide a uniform current density throughout the microchannel.

[0018] 3A-3C provide two configurations of microchannels containing electrodes and show the current generation in each simulated microchannel configuration.

[0019] 4A-B are schematic diagrams showing simulated current generation and thrombus detection in two different configurations: Figure 4A shows an interdigitated configuration and Figure 4B shows a two-electrode configuration.

[0020] Figures 5A-5F provide thrombus detection data for the six configurations described in Example 4. In each graph, impedance (Ohms, Y-axis) is plotted against time (Seconds, X-axis).

[0021] 6A-6B provide simulations of current flow in microchannels of various channel heights using a two-electrode microchannel configuration, showing how the current distribution becomes less uniform as the microchannel height increases.

[0022] Microchannel devices with electrodes can be used for biological fluid analysis as well as environmental analysis such as water quality testing. The device may include a sensor separate from the electrodes or the electrodes themselves may act as sensors, in either case the sensor detects electrical changes in the channel medium (e.g., changes in one or more electrical properties such as admittance, conductivity, impedance, resistance, permittivity, etc.) that occur, for example, as a result of a chemical reaction or physical change in the medium, or both. The detection and measurement of electrical changes in a sample is sometimes referred to as impedance spectroscopy or dielectric spectroscopy. When assessing clotting of a sample, impedance spectroscopy may involve evaluating changes in impedance over a range of frequencies, for example when examining whole blood containing both plasma and blood cells.

[0023] In certain embodiments described herein, the electrodes act as sensors that measure changes in electrical properties in the microchannel medium.

[0024] In some embodiments, the electrodes in the microchannels may serve additional functions. For example, in certain cases involving chemical reactions, the electrodes themselves may serve to initiate the chemical reaction. In some cases, the electrodes may include materials that participate in the chemical reaction (e.g., in the form of a coating on the electrodes, etc.).

[0025] Additionally, the electrodes may be functionalized with one or more types of agents (substances). Such agents may play several roles, such as participating in a chemical reaction, localizing the reaction to the sensor area, or both. For example, electrodes may be functionalized with proteins such as antibodies or antigens for the purpose of direct or indirect ELISA.

[0026] Electrodes in a microchannel may also promote mixing of the medium with other contents in the microchannel. For example, the electrodes may disrupt the flow of the medium to promote mixing by providing a physical obstacle to the flow of the medium in the microchannel. See, in their entirety, Nguyen et al., Recent Advances and Future Perspectives on Microfluidic Liquid Handling, Micromachines 8: 186 (2017), and Ward and Fan, Mixing in Microfluidic Devices and Methods for Promoting It, Micromech. Microeng., 25, 094001 (2015). Additionally or alternatively, an applied voltage may provide a mixing effect. See, for example, Song et al., Chaotic Mixing in Microchannels via Low-Frequency Switched Transverse Electroosmotic Flow Generated on Integrated Microelectrodes, Lab on a Chip, 10:734-740 (2010). In certain embodiments, a lower ratio of electrode height:channel height may be preferred depending on the mechanism of mixing desired.

[0027] Microfluidic channels with electrodes can be used to measure a variety of changes in blood or in one or more components of blood. In embodiments of the present invention, a microchannel-electrode configuration is provided that is particularly useful for detecting and assessing coagulation, a process that results in the formation of a cross-linked fibrin clot. Additionally, in the case of whole blood, the devices and methods described herein can be used to detect and / or measure changes in circulating cells, such as changes in capacitance of red blood cell membranes. In other embodiments, the devices and methods can be used to detect and / or measure changes in platelet morphology and activation (e.g., adhesion and aggregation).

[0028] For example, measuring changes in electrical impedance (or one or more other electrical properties such as resistance, capacitance, inductance, and dielectric constant) may be used for thrombus detection in a microfluidic channel. In some examples, electrodes may be disposed on a glass or other substrate that activates the coagulation contact pathway. Placing electrodes on a coagulation contact pathway activating substrate (e.g., glass) may promote localization of thrombus formation on such substrate, i.e., in the vicinity where the electrode is disposed, and promoting the formation of thrombus around the electrode may enhance thrombus detection by the electrode. Thus, in certain embodiments, electrodes may be disposed directly on a thrombotic substrate.

[0029] The substrate on which the electrodes are disposed may be the same as or different from the material (or materials) that form the surface of the microchannel. For example, in some embodiments, the electrodes are disposed directly on the floor of the microchannel, and the floor of the microchannel is the substrate on which the electrodes are disposed, while in other embodiments there is an additional material (different from the floor of the microchannel) that serves as the substrate on which the electrodes are disposed.

[0030] In some embodiments, the electrodes are mounted on a minimally thrombogenic substrate (such as, for example, Kapton®), with the electrodes themselves being the most thrombogenic surface in the microchannel. Similarly, in certain embodiments in which the substrate on which the electrodes are mounted is also thrombogenic, the electrodes are more thrombogenic than the substrate (and all other surfaces in the microchannel), thereby making the electrodes the most thrombogenic surface in the microchannel. In such embodiments in which the electrodes provide the most thrombogenic surface in contact with the sample, the electrodes help localize thrombus formation in the vicinity of the sensor (or on the sensor, in embodiments in which the electrodes also act as sensors), which can further improve the device's ability to detect thrombus formation. Using such devices to assess clotting can be done without the addition of an activator, as the electrodes act as activators of the intrinsic clotting pathway. Alternatively, activators (e.g., glass microspheres or powdered glass, kaolin, silica, activated clotting factors, phospholipids, tissue factor, etc.) may be added. The electrodes can be made thrombogenic, for example, by including a coating (gold or other metal coating) on ​​the electrodes. See Hulander et al., Blood Interactions with Precious Metals: Coagulation and Immune Complement Activation, ACS Appl. Mater Interfaces 2009 May;1(5):1053-62 in its entirety.

[0031] Few studies have described clotting detection in microchannels made entirely or partially of materials that are minimally thrombogenic (e.g., materials such as various plastics, including but not limited to polyimide). One example is the use of planar electrodes placed on the parallel sides of the channel; see, for example, Kucukal et al., Monitoring Blood Clotting Using Surface-Functionalized Microfluidic Dielectric Sensors, 2017 IEEE 12th International Conference on Nano / Micro Engineered and Molecular Systems (NEMS), 752(755 (polyethylene glycol-coated microchannel), and Maji et al., Assessment of Whole Blood Clotting Using Microfluidic Dielectric Sensors, J. Thromb. Haemost. 16:2050(2056 (2018) (polymethyl methacrylate (PMMA) plastic substrate). In another example, electrodes are placed in their own channel (separate from the channel containing the sample) and the current must pass through the microtube to detect thrombus formation.

[0032] An embodiment of the present invention provides a microchannel having electrodes in a monoplanar configuration. The use of monoplanar electrodes provides several advantages. For example, the use of monoplanar electrodes (as opposed to multiplanar) may allow for the use of a greater variety of manufacturing techniques and materials, including more ways to incorporate electrodes into a microchannel. In particular, the use of monoplanar electrodes allows for the use of a printed circuit board (PCB) as the substrate on which the monoplanar electrodes are mounted, thereby allowing for the use of a PCB to form one or more microchannel surfaces.

[0033] In further embodiments, the monoplanar electrodes may be mounted on a minimally thrombogenic substrate. Using a minimally thrombogenic substrate may provide similar advantages for fabricating the microchannel-electrode system. For example, with respect to using a PCB, the PCB may be made of a minimally thrombogenic material, such as polyimide. Minimally thrombogenic materials include materials that generally have a low tendency to produce clots when in contact with blood, as compared to other materials that are considered thrombogenic (e.g., materials such as glass and gold). Minimally thrombogenic materials include materials that may be referred to as non-thrombogenic. It should be understood that there are various techniques that may be used to mount the electrodes on the substrate, such as, for example, plating, printing, electrospray deposition, sputtering, annealing, and gluing (by adhesive or other methods).

[0034] In certain embodiments, the materials used for the surface of the substrate and the materials used for the interior surface of the microchannel (if different from the substrate or substrate material) are minimally conductive (low conductive materials also include materials that are called non-conductive). The conductivity of a material refers to the ability of the material to conduct electric current. In the embodiments described herein, the materials that contact the sample in the microchannel (e.g., the materials used for the inner surface of the microchannel) have low electrical conductivity compared to the conductivity of the sample, such that negligible amounts of electric current flow through the material. Thus, minimally conductive materials include, for example, materials that are poorly conductive compared to materials such as gold and blood (e.g., polyimide, polycarbonate, and acrylic, etc.) (see Table 2).

[0035] Thus, in some embodiments, the present invention provides a single planar electrode in a single microfluidic channel. Such embodiments further provide a microchannel-electrode configuration that produces a uniform current distribution. In the examples described herein, the use of a single planar electrode disposed on a minimally thrombogenic and minimally conductive substrate (e.g., a substrate having or coated with a plastic such as polyimide) produces a uniform current density throughout the microchannel, allowing successful detection of clot formation throughout the microchannel (as opposed to only at specific locations or portions within the microchannel). Although some of the examples described herein relate to detection of clotting in a sample, the microchannels described herein are useful for any application in which a uniform current flow within the microchannel is desirable (e.g., aggregation and / or coagulation measurements). For example, the various microchannel-electrode configurations described herein can be used not only to treat a sample with an electric current, but also for any impedance measurement (or conductivity measurement, etc.) analysis of the sample (e.g., detection of clotting, but also detection of other changes in the sample that affect electrical properties, etc.).

[0036] In addition to providing uniform current distribution, the microchannels described herein can be used for small sample volumes due to the small microchannel volume of 2.0 (2.5 μL). Also, because the microchannels described herein are single-planar electrodes, they are less complicated to manufacture than other devices that use multi-planar electrodes. For example, the microchannel configurations shown in FIG. 3B and FIG. 6 can be fabricated using PCBs.

[0037] As described herein, having a uniform current density throughout a microchannel, or having a uniform current distribution throughout a microchannel, means having current that is generally uniformly distributed throughout the microchannel with few or no cold spots where the current density is substantially lower than the surrounding areas.

[0038] The present invention provides configurations of electrodes with respect to microchannel geometries to achieve the advantages described herein. The relationship between electrodes and microchannel geometries can be critical, especially when the goal is to detect thrombus formation on a minimally thrombogenic and minimally conductive substrate (which in the examples described herein is the floor of the microchannel). For example, when evaluating current distribution within various microchannel-electrode configurations as shown herein, the current distribution, and therefore the ability to detect changes in current within the microchannel, is biased based on the location of the electrodes. For example, as shown in Figures 2B and 4A, which show an interdigitated electrode configuration, when a voltage is applied between the middle electrode and each of the outer electrodes, current flows between the middle electrode and both outer electrodes, but not to other regions of the microchannel. Another example is the configuration shown in Figure 2C, which has six electrodes (one electrode in the center of a hexagon, one electrode spanning each side of the hexagon). In simulations of this configuration, voltage was applied between the middle electrode and one of the six outer electrodes at each time. Simulations of this configuration show how current preferentially flows to the nearest outer electrode (which is the nearest conductive material) rather than the center electrode. With such a configuration, thrombi formation in areas of the channel with relatively low current density (e.g., blue and purple areas shown in the figure) results in poor thrombi detection resolution and poor thrombi detection sensitivity (see, e.g., FIG. 4).

[0039] In addition, in a hexagonal two-electrode microchannel configuration, for example, the electrodes may have a uniform current distribution between the electrodes, but if the microchannel extends laterally or dorsally beyond the electrodes, such extension may reduce the uniformity of the current density within the microchannel. The examples provided herein illustrate this possible outcome. In Example 4, electrical impedance thrombus detection was performed on various configurations of a hexagonal two-electrode microchannel (see Table 1 and FIG. 5). In general, an electrode:channel height ratio greater than 0.5 allows for sensitive thrombus detection (see, for example, configurations 1 and 2). In Example 5, current distribution simulations were performed using various channel heights for the configuration shown in FIG. 6A. When the electrode:channel height ratio was 0.0875, the current density was relatively uniformly distributed (see FIG. 6B(ii)), while when the electrode:channel height ratio was 0.35, a uniform current density distribution was obtained at a relatively high density level (see FIG. 6B(i)).

[0040] The current distribution within the microchannel can also be affected by the distance between the electrodes relative to the width of the microchannel: spacing the electrodes closer together and further from the ends of the channel can, in some cases, result in poorer detection of thrombi (see, for example, Figure 5, Configuration 5 and Table 1).

[0041] As demonstrated herein, by using specific microchannel geometries and electrode configurations with appropriate electrode:channel height ratios and electrode spacing, uniform current distribution was achieved within the microchannels, enabling robust thrombus detection even when minimally thrombogenic substrates were used for the electrode and microchannel surfaces.

[0042] The microchannel-electrode configurations provided herein can be used in applications other than assessing blood clotting, and are useful in any application where it is desired to detect or measure a change in an electrical property of a material within a microchannel. The configurations can also be used to generate a uniform current in a material within a microchannel, even when it is not necessary to detect or measure a change in an electrical property. For example, in certain scenarios, it may be desirable to apply a current to a material and to have the current flow uniformly across the material. The present invention provides a microchannel-electrode configuration having a single planar electrode that allows for such uniform application of current.

[0043] Although the present invention provides a microchannel-electrode system that does not require a thrombogenic substrate to successfully detect thrombus formation, it should be understood that a thrombogenic substrate may be used in any of the various embodiments of the microchannel-electrode configurations described herein. A thrombogenic substrate has surface properties that activate clotting when the material comes into contact with blood. Examples of thrombogenic substrates include, for example, glass and metals such as gold.

[0044] Additionally, in some embodiments, the electrodes can have a thrombogenic material. For example, in certain embodiments, the electrodes have a coating with a thrombogenic material or are otherwise made of a material having a thrombogenic material. Thrombogenic materials that can be used to coat the electrodes include non-metals such as collagen, as well as metals, e.g., gold, silver, platinum, and other metals that stimulate clotting when in contact with blood. The use of electrodes having a thrombogenic material can help localize thrombus formation to the electrode area, increasing the sensitivity of thrombus detection.

[0045] The following examples are intended only to illustrate the invention and its embodiments, and are not to be construed as limiting the scope or spirit of the invention. EXAMPLES

[0046] Example 1: Simulations of current generation were performed for various microchannel-electrode configurations, as shown in Figures 2A-2D. The simulations yielded different current density distributions for each configuration. However, none of them yielded uniform current distribution throughout the microchannel. The configuration shown in Figure 2B shows that the current density in the microchannel is relatively uniform, but the current is concentrated at both poles and at the center of each electrode. Simulations were performed using COMSOL Multiphysics 5.4 with the AC / DC Module (COMSOL, Inc., Burlington, MA, USA). The properties of the medium in the microchannel were similar to unclotted blood (electrical conductivity: 0.703 S / m, relative permittivity: 5120).

[0047] Figure 2A shows the interdigitated morphology. The excitation voltage was 0.6 V AC between each of the two outer electrodes and the middle electrode. Figure 2B shows the two-electrode morphology. The excitation voltage was 0.6 V AC between the two electrodes. Figure 2C shows the hexagonal multiplexed morphology. An excitation voltage of 0.6 V AC was applied between the center electrode and one of the six edge electrodes for a fixed period of time. This morphology reflects an attempt to improve the spatial resolution of thrombus detection in the microchannel by measuring a small portion of the microchannel at a time. However, the inert electrodes directed the current away from the media, reducing the uniformity of the current density. Figure 2D shows the multiplexed morphology with three electrode pairs. In another attempt to improve the spatial resolution of detecting current changes, an excitation voltage of 0.6 V AC was applied between one of the three electrode pairs in the microchannel. However, this morphology, similar to the morphology in Figure 2C, had poor uniformity of the current density.

[0048] For all figures showing current density (Fig. 2A(ii)(v), Fig. 2B(ii)(iii), Fig. 2C(ii)(iii), Fig. 2D(ii)-(iv), Fig. 3A(ii)(iii), Fig. 3B(ii)(iii), Fig. 4A(ii), Fig. 4B(ii), and Fig. 6B(i)(v)), the two vertical legends to the right of the microchannel diagram indicate the current density (A / m 2 ) The first (left) vertical legend gives the values ​​for the contour plot, and the second (right) vertical legend gives the values ​​for the surface plot.

[0049] Example 2: Simulated current generation was compared for microchannels with the same geometry but different electrode morphologies, as shown in Figures 3A-3C. The electrodes in Figures 3A and 3B have the same height (35 μm) and width (100 μm), but the location of the electrical vias was changed in Figure 3B (relative to the location in Figure 3A). Additionally, each electrode in Figure 3B has two circular or valve regions, one at each electrode end, whereas each electrode in Figure 3A has one circular region (containing the vias and located at the center of the electrode). In Figure 3B, the circular regions at each electrode end are 0.5 mm in diameter (see also Figure 3C). These differences in electrode geometry significantly affected the current distribution in the microchannels (compare Figure 3A(ii) and Figure 3B(ii)). By varying the position, size, and number of the circular regions of each electrode in the microchannel, the uniformity of the current in the microchannel is improved, as shown in Figure 3B, and the current density level is generally higher (e.g., compare the current density levels in the central region of Figure 3A(ii) and Figure 3B(ii)). Comparing these morphologies shows how changing the electrode geometry, such as the poles of the electrodes, can affect the current distribution in the microchannel. The simulations were performed using COMSOL Multiphysics 5.4 with the AC / DC Module (COMSOL, Inc., Burlington, MA, USA). The properties of the medium in the microchannel were approximated to unclotted blood (conductivity: 0.703 S / m, relative permittivity: 5120). The excitation voltage was 0.6 V AC between the two electrodes in all cases.

[0050] Example 3: The simulated current density was compared for two microchannel-electrode configurations, as shown in Figures 4A-4B. Figure 4A shows the interdigitated configuration, and Figure 4B shows the two-electrode configuration. The simulations were performed using COMSOL Multiphysics 5.4 with the AC / DC Module (COMSOL, Inc., Burlington, MA, USA). The properties of the medium in the microchannel were similar to non-clotting blood (conductivity: 0.703 S / m, relative permittivity: 5120). The excitation voltage was 0.6 V AC between the two outer and middle electrodes in Figure 4A (interdigitated configuration) and between the two electrodes in Figure 4B (two-electrode configuration). The schematics in Figures 4A(iii) and 4B(iii) show how a more uniform current distribution improves thrombus detection. In Figure 4A(iii), thrombi (represented by a collection of white circles) in the blue regions (regions of low current density) are difficult to detect; to detect a thrombi in this morphology, the thrombi must be large enough to overlap with the green regions of higher current, and thus thrombi detection depends on the location of the thrombi in the microchannel and the size of the thrombi. In contrast, the more uniform current distribution in Figure 4B(iii) increases the chances of successfully detecting early thrombus formation in the microchannel, since detection is less dependent on the location of the thrombus formation.

[0051] Example 4: Six configurations were evaluated for their ability to detect thrombus formation based on changes in electrical impedance. The configurations are shown in FIG. 5 and listed in Table 1. The electrodes used in each configuration were gold plated electrodes. Each electrode contained one circular via (connection to the other side of the circuit board that interfaces with external electronics) located at one pole (end) of the electrode. The circular via is surrounded by a circular area or bulb in the electrode. In certain configurations, a circular shape was mimicked at the other end of the electrode, see configurations 1, 2, 3, and 6 in FIG. 5. Using an electrode with a circular shape at both electrode ends promotes symmetrical current distribution, but using an electrode with a circular shape at only one end may result in a colder spot toward the end without the circular shape because that portion of the electrode is farther away from the opposing electrode than the electrode end with the circular shape.

[0052] A thrombus is detected when the impedance slope changes from negative to positive. In Table 1, the time corresponding to this change in slope is provided as the “thrombus detection time.” As the cross-linked fibrin clot continues to form, the electrical impedance increases until it reaches a peak. The graph in FIG. 5 shows how the electrode geometry and placement and the microchannel geometry affect the ability of the electrodes to sense the change in electrical impedance to detect thrombus formation. Although all configurations were successful in detecting thrombus, the thrombus detection time, which is an indicator of the sensitivity of the microchannel-electrode system, varies between configurations. For example, configuration 2 (FIG. 5B) detects a thrombus at 300 seconds, while configuration 3 (FIG. 5C) does not detect a thrombus until about 480 seconds. An additional parameter is the change in impedance that occurs when a thrombus forms; the greater the increase in impedance starting from when the impedance slope changes from negative to positive, the more sensitive the thrombus detection, which may be due to increased sensitivity to the change in cell membrane capacitance associated with clotting, improving detection capabilities. For example, Configuration 1 (FIG. 5A) exhibited a strong thrombus detection signal with an impedance change of over 200 ohms, while Configuration 6 (FIG. 5F) exhibited a relatively poor thrombus signal with an impedance change of less than 100 ohms. Configuration 2 (FIG. 5B) also exhibited sensitive thrombus detection.

[0053] Example 5: Simulations of current generation were performed in a two-electrode configuration to evaluate the effect of channel height on the current distribution, as shown in Figures 6A-6B. Simulations were performed using COMSOL Multiphysics 5.4 with the AC / DC Module (COMSOL, Inc., Burlington, MA, USA). The properties of the medium in the microchannel were similar to non-clotting blood (conductivity: 0.703 S / m, relative permittivity: 5120). The excitation voltage was 0.6 V AC between the two electrodes at a frequency of 10 kHz. The current density (A / m) in the medium along the indicator surface (see Figure 6A(iii)) was measured for microchannels spanning different heights from 100 μm to 2400 μm (100, 200, 300, 400, 600, 800, 1200, 1600, 2400 μm).2 ) was plotted. All other channel dimensions were kept constant and are shown in Figure 6A(ii) (length = 6 mm, ends = 4 mm, width = 5 mm, electrode spacing = 4.5 mm). The electrode height was 35 μm. The electrode spacing (4.5 mm) is the spacing of the straight portions of the electrodes excluding the rounded ends. Figure 6B is a plot of the current density for microchannel heights of 100 μm, 400 μm, 1,200 μm, 1,600 μm, and 2,400 μm. As the microchannel height increases, the current distribution becomes less uniform. With increasing microchannel height, the current is given more volume to spread out, which reduces the current density the further from the electrodes, and overall reduces the uniformity of the current density.

[0054] Furthermore, excitation frequencies ranging from 100 Hz to 1 MHz were used in the simulations. Varying the frequency had a negligible effect on the current distribution, indicating that the impedance between the electrodes is primarily resistive in this model. In Figure 6B, a frequency of 10 kHz is shown for all cases.

[0055] Example of a microchannel with two electrodes [Table 1]

[0056] conductivity [Table 2]

[0057] Unless expressly specified, numerical ranges and values ​​described herein, such as dimensional ratio values, may be read as being prefaced by the word "about" even if the term "about" is not expressly stated in conjunction with the numerical range or value. At the very least, and without any attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical value should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques. Furthermore, when numerical ranges are set forth herein, these ranges include the endpoints of the ranges recited (e.g., endpoints may be used).

[0058] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art in light of this disclosure that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A microchannel having two electrodes disposed on a single plane of the microchannel, wherein the shape of the microchannel is a hexagon having six sides, each of the two electrodes has a rectangular region, and the rectangular region of each electrode is parallel to one of the six sides of the microchannel and parallel to the rectangular region of the other electrode, The following features a. The ratio of the electrode height to the channel height is at least 0.1 b. The ratio of the electrode spacing to the channel width is at least 0.5 c. Each of the two electrodes has two circular regions, and each circular region is located at the electrode end A microchannel in which one or more of the above exist.

2. The ratio of the electrode height to the channel height is 0.3 or more, The microchannel according to Claim 1.

3. The ratio of the electrode height to the channel height is 0.5 or more, The microchannel according to Claim 1.

4. The ratio of the electrode height to the channel height is 0.6 or more, The microchannel according to Claim 1.

5. The ratio of the electrode height to the channel height is 0.8 or more, The microchannel according to Claim 1.

6. Each of the two electrodes has two circular regions, each circular region is located at the electrode end, and each circular region has a diameter of 500 microns, The microchannel according to Claim 1.

7. Each of the two electrodes has two circular regions, each circular region is located at the electrode end, and each circular region has a diameter in the range of 300 microns to 600 microns, The microchannel according to Claim 1.

8. Each circular region has a diameter of 600 microns, The microchannel according to claim 7.

9. The volume of the microchannel is 2.5 microliters or less, The microchannel according to any one of claims 1 to 8.

10. The volume of the microchannel is 2.0 microliters or less, The microchannel according to any one of claims 1 to 8.

11. The volume of the microchannel is 2.0 to 2.5 microliters, The microchannel according to any one of claims 1 to 8.

12. The inner surface of the microchannel has minimal thrombogenicity, The microchannel according to claim 9.

13. The inner surface of the microchannel has minimal conductivity, The microchannel according to claim 9.

14. Each of the two electrodes provides a surface that is more thrombogenic than all other surfaces within the microchannel, The microchannel according to claim 9.

15. The floor of the microchannel has polyimide, The microchannel according to claim 9.

16. Each of the two electrodes has a thrombogenic coating, The microchannel according to claim 9.

17. The thrombogenic coating has one or more of gold, silver, and platinum, The microchannel according to claim 16.

18. The ratio of electrode height to channel height is at least 0.1, the ratio of electrode spacing to channel width is at least 0.5, each of the two electrodes has two circular regions, and each circular region is located at the electrode end. The microchannel according to claim 9.

19. A hexagonal microchannel having two electrodes oriented to face each other and on the same plane of the hexagonal microchannel, each electrode having a rectangular region that is parallel to one of the six sides of the hexagonal microchannel and is located at a position of 500 microns or less from the parallel side, the distance between the rectangular regions of the electrodes being at least 4,000 microns, and the length of each side being 4,000 microns or less.

20. The ratio of electrode height to channel height is 0.3 or more. The hexagonal microchannel according to claim 19.

21. The inner surface of the hexagonal microchannel has minimal thrombogenicity. The hexagonal microchannel according to any one of claims 19 to 20.

22. The inner surface of the hexagonal microchannel has minimal conductivity. The hexagonal microchannel according to claim 19 or 20.

23. The floor of the hexagonal microchannel has polyimide. The hexagonal microchannel according to claim 19 or 20.

24. Each of the two electrodes has a thrombogenic coating. The hexagonal microchannel according to claim 19 or 20.

25. The thrombogenic coating has one or more of gold, silver, and platinum. The hexagonal microchannel according to claim 24.

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