Fluid control in microfluidic devices

Abstract

A diagnostic system for determining the presence of a target in a sample liquid includes a diagnostic reader and a microfluidic strip having a microfluidic channel network therein. An actuator in the reader varies the pressure of a gas in gas communication with a liquid-gas interface of the sample liquid in the microfluidic channel network to move and / or mix the sample liquid. The pressure variation can be continuous and / or oscillatory.

Description

Background Art

[0001] [Technical Field] The present invention relates to the manipulation of liquids within a microfluidic device.

[0002] [Cross - Reference to Related Applications] This application claims the benefit and priority of U.S. Patent Application No. 62 / 960,421, filed on January 13, 2020; U.S. Patent Application No. 62 / 972,921, filed on February 11, 2020; U.S. Patent Application No. 62 / 991,446, filed on March 18, 2020; U.S. Patent Application No. 63 / 032,410, filed on May 29, 2020; U.S. Patent Application No. 63 / 055,744, filed on July 23, 2020; U.S. Patent Application No. 63 / 067,782, filed on August 19, 2020; and U.S. Patent Application No. 63 / 092,371, filed on October 15, 2020. The entire disclosure of each of these applications is hereby incorporated by reference in its entirety herein.

[0003] [Background Art] A cartridge (e.g., a strip) having a microfluidic channel network can be used, for example, to determine the presence or amount of one or more targets in a sample liquid and / or to determine the physiological characteristics of the sample liquid. Such a cartridge can be used with a reader. The reader operates the cartridge to perform fluidic and / or detection functions, for example, in determining a target or in determining the physiological, physiochemical, or other characteristics of a sample.

[0004] Manipulation of the sample and / or other liquids within the cartridge is often performed to ensure, for example, that a reagent deposited within or introduced into the cartridge contacts, mixes with, and / or reacts with the sample.

Summary of the Invention

[0005] In some embodiments, the present invention relates to a method of manipulating a liquid disposed within a capillary channel and having a liquid-gas interface, the method including the step of vibrating the gas pressure of the gas at the liquid-gas interface. The vibration can induce mixing of materials within the liquid. The materials can include, for example, a target compound or other material of interest present within the liquid introduced into the capillary channel and / or a reagent or other material contacted by the liquid within the capillary channel.

[0006] In some embodiments of the method of manipulating a liquid, the capillary channel can include a proximal origin and a distal end. The liquid is introduced into the capillary channel via application to the proximal origin. The liquid-gas interface of the liquid can be the liquid-gas interface on the distal side of the liquid disposed between the proximal origin and the distal end of the capillary channel, and the gas of the distal-side liquid-gas interface occupies at least the distal end of the capillary channel.

[0007] In some embodiments of the method of manipulating a liquid, the step of vibrating the gas pressure is performed by vibrating the gas pressure peak-to-peak by an overall relative amount that is at least about 5%, at least about 10%, at least about 20%, at least about 25%, or at least about 35%, where ((P max -P min ) / P avg )×100). Here, P max is the maximum gas pressure during the vibration cycle, P min is the minimum gas pressure during the vibration cycle, and P avg is the average gas pressure during the vibration cycle. The step of vibrating the gas pressure of the gas is an overall relative amount that is about 300% or less, about 200% or less, about 135% or less, about 100% or less, or about 75%, where ((P max -P min ) / P avg)×100) peak-to-peak. The step of oscillating the gas pressure of the gas may be performed by oscillating the gas pressure by a total amount (P) that is at least about 5 kPa, at least about 10 kPa, at least about 20 kPa, at least about 25 kPa, or at least about 35 kPa. max -P min The step of oscillating the gas pressure of the gas may be performed by oscillating the gas pressure peak-to-peak by a total amount (P) of about 200 kPa or less, about 135 kPa or less, about 100 kPa or less, or about 75 kPa or less. max -P min This can be done by oscillating the gas pressure peak-to-peak by

[0008] In some embodiments of the method of manipulating the liquid, the step of oscillating the gas pressure may include the step of oscillating the volume occupied by the gas within the capillary channel, such as within the distal end portion of the capillary channel. For example, the step of oscillating the gas pressure may be performed by oscillating at least a portion of the wall of the capillary channel peak-to-peak over a total distance that is at least about 5 μm, at least about 7.5 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, or at least about 30 μm. The step of oscillating the gas pressure may be performed by oscillating at least a portion of the wall of the capillary channel peak-to-peak over a total distance that is about 70 μm or less, about 60 μm or less, about 50 μm or less, or about 40 μm or less. The total distance may be at least about 5%, at least about 7.5%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% of the total dimension of the capillary channel, such as the height, along an axis aligned with the oscillatory motion of the wall, peak-to-peak. The total distance may be about 70% or less, about 60% or less, about 50% or less, or about 40% of the total dimension of the capillary channel, such as the height, along an axis aligned with the oscillatory motion of the wall, peak-to-peak. The distance (length) and direction of the dimensions of the capillary channel may be grasped (measured) along an axis that is substantially perpendicular to the longitudinal axis of the capillary channel at the location of the oscillation of the wall and / or substantially perpendicular to the plane containing the capillary channel.

[0009] In some embodiments of the method of manipulating the liquid, the step of vibrating the gas pressure can be performed by vibrating at least a portion of the wall of the capillary channel. The method can include the step of placing at least a portion of the wall under tension before starting the step of vibrating at least a portion of the wall. At least a portion of the wall is in direct communication with the gas, for example, directly above or below the gas, but may not be in direct communication with the liquid, for example, not directly above or below the liquid. For example, a portion of the wall of the capillary channel that is vibrated and in direct communication with the gas can be located, for example, at least about 0.2 cm, at least about 0.3 cm, at least about 0.5 cm, at least about 0.75 cm, at least about 1.00 cm, at least about 1.25 cm, or at least about 1.5 cm away from the liquid-gas interface of the liquid, for example, the distal liquid-gas interface, along the longitudinal axis of the capillary channel. The step of vibrating at least a portion of the wall of the capillary channel can be performed by subjecting the wall of the capillary channel, for example, the wall of the distal end of the capillary channel, to repeated cycles of deformation and relaxation. The volume occupied by the gas can decrease when increasing the deformation of the wall of the capillary channel and can increase when increasing the relaxation of the wall of the capillary channel. In the undeformed state, the outer surface of the wall is substantially planar. In the deformed state, the outer surface of the wall can be concave and can become more concave as the deformation increases, while the inner surface of the wall can be convex and can become more convex as the deformation increases. Increasing the deformation of the wall can increase the tension received by the wall, and decreasing the deformation of the wall can decrease the tension received by the wall.

[0010] In some embodiments of the method of manipulating the liquid, the capillary channel can seal the gas during vibration, except for the passage (movement) of gas towards and away from the liquid-gas interface along the capillary channel. For example, the gas during vibration can occupy the distal end portion of the capillary channel, and the distal end portion of the capillary channel can be sealed with respect to the inflow (invasion) and outflow (leakage) of gas, except for the passage (movement) of gas towards and away from the liquid-gas interface along the capillary channel.

[0011] In some embodiments of the method of manipulating a liquid, the liquid-gas interface can be a first liquid-gas interface, and the liquid disposed within the capillary channel can have a plurality of second liquid-gas interfaces. A first set of the plurality of second liquid-gas interfaces can be disposed along a first sidewall of the capillary channel. A second set of the plurality of second liquid-gas interfaces can be disposed along a second sidewall of the capillary channel, and the second sidewall can be opposite (facing) the first sidewall. The first liquid-gas interface can have an axis of symmetry that is substantially aligned with the longitudinal axis of the capillary channel at the location of the first liquid-gas interface, and each of the second liquid-gas interfaces can have an axis of symmetry at a non-zero angle with respect to the axis of symmetry of the first liquid-gas interface and / or at a non-zero angle with respect to the longitudinal axis of the capillary channel at the location of such second liquid-gas interface. The non-zero angle can be at least about 20°, at least about 35°, at least about 45°, at least about 67.5°, or at least about 85°. The non-zero angle can be about 160° or less, about 145° or less, about 112.5° or less, or about 95° or less. For example, the axes of symmetry of the first and second liquid-gas interfaces can be substantially perpendicular to each other. Alternatively, the axis of symmetry of each of the first set of second liquid-gas interfaces can be oriented at a first angle with respect to the longitudinal axis of the capillary channel, and the axis of symmetry of each of the second set of second liquid-gas interfaces can be oriented at a second different angle with respect to the longitudinal axis of the capillary channel. The first and second angles can be opposite (facing) each other. For example, the axis of symmetry of each of the first set of second liquid-gas interfaces can be oriented generally proximally along the capillary channel, and the axis of symmetry of each of the second set of second liquid-gas interfaces can be oriented generally distally along the capillary channel.

[0012] In some embodiments of the method for manipulating a liquid, the capillary channel may include one or more openings disposed along its first sidewall, where the liquid contacts the gas at each of the one or more openings to form a second liquid-gas interface thereat, e.g., adjacent to the first sidewall of the capillary channel. The capillary channel may include one or more openings disposed along its second sidewall, where the liquid contacts the gas at each of the one or more openings in the second sidewall to form a second liquid-gas interface thereat, e.g., adjacent to the second sidewall of the capillary channel. The first and second sidewalls may be opposite each other (facing each other). Each of the openings in the first and / or second sidewalls may be an opening to a cavity containing the gas at the second liquid-gas interface. Each of the one or more cavities may have a longitudinal axis that, at the location of the cavity's opening to the capillary channel, is angled at least about 20°, at least about 35°, at least about 45°, at least about 67.5°, or at least about 85° relative to the longitudinal axis of the capillary channel. Each of the one or more cavities may have a longitudinal axis that, at the location of the cavity's opening to the capillary channel, is angled at no more than about 160°, no more than about 145°, no more than about 112.5°, or no more than about 95° relative to the longitudinal axis of the capillary channel. For example, the longitudinal axis of each of the plurality of cavities and the longitudinal axis of the capillary channel at the location of the cavity's opening to the capillary channel may be approximately perpendicular to each other. In some embodiments, the longitudinal axis of each of the first set of cavities is oriented at a first angle relative to the longitudinal axis of the capillary channel, and the longitudinal axis of each of the second set of cavities is oriented at a second angle relative to the longitudinal axis of the capillary channel, the first and second angles being opposite each other. For example, the opening of each of the first set of cavities can face generally proximally within the capillary channel, and the opening of each of the second set of cavities can face generally distally within the capillary channel.

[0013] In some embodiments of the method of manipulating a liquid containing a second liquid-gas interface, the second liquid-gas interface is configured and arranged such that the net effect of oscillating the gas pressure of the gas at the first liquid-gas interface, for example, the net effect of oscillating the gas pressure at an acoustic frequency, induces little or no resultant force (net force), for example, tends not to induce a substantial resultant force and induce a bulk movement of the first liquid-gas interface along the longitudinal axis of the capillary channel.

[0014] In some embodiments of the method of manipulating a liquid, the oscillating step may be performed at an acoustic frequency, for example, about 15,000 Hz or less, about 10,000 Hz or less, about 5,000 Hz or less, about 3,000 Hz or less, about 2,000 Hz or less, about 1,750 Hz or less, about 1,500 Hz or less, about 1,250 Hz or less, about 1,150 Hz or less, about 1,050 Hz or less, or about 950 Hz or less. The oscillating step may be performed at about 25 Hz or more, about 50 Hz or more, about 100 Hz or more, about 150 Hz or more, about 200 Hz or more, about 250 Hz or more, about 500 Hz or more, about 750 Hz or more, or about 900 Hz or more.

[0015] In some embodiments of the method of manipulating a liquid, the oscillating step may be performed during a time T osc . In some embodiments, the time T osc is at least about 1 second, at least about 2 seconds, at least about 5 seconds, at least about 15 seconds, or at least about 20 seconds. In some embodiments, the time T osc is about 180 seconds, about 120 seconds or less, about 90 seconds or less, about 45 seconds or less, or about 30 seconds or less.

[0016] The oscillating step may be performed at an essentially constant frequency during the time T osc . The oscillating step may vary the oscillation frequency during the time T osc by increasing or decreasing the oscillation frequency as, for example, a linear or non-linear ramp, and / or by periodically changing the oscillation frequency during the time T osccan be executed at a frequency that varies between. The oscillation frequency is the time T osc can be varied over the entire range of at least about 2.5%, at least about 5%, at least about 7.5%, or at least about 10% of the average frequency during. The oscillation frequency is the time T osc can be varied over the range of about 30% or less, about 25% or less, about 20% or less, or about 15% or less of the average frequency during. The oscillation frequency may be smooth, may be stepped, for example, stepped at about 2.5 Hz, about 5 Hz, about 7.5 Hz, or about 10 Hz. The time to vary the oscillation frequency over the entire range of frequency variation, for example, the time T osc the time of periodic variation within is the time T osc can be at least about 1%, at least about 2%, at least about 2.5%, at least about 3.5%, or at least about 5% of. The time to vary the oscillation frequency over the entire range of frequency variation is the time T osc can be at least about 10% or less, about 15% or less, about 10% or less, about 7.5% or less, or about 5% or less of. For example, for a time T of about 25 seconds osc the average oscillation frequency of is about 1100 Hz, and the oscillation frequency can be varied as a triangular wave between about 1050 Hz and about 1100 Hz during the time T osc and the triangular wave can have a period of about 2 seconds.

[0017] In combination, or alternatively, the oscillation step can be executed with an essentially constant peak-to-peak displacement during the time T osc The oscillation step can be executed, for example, with a linear or non-linear ramp to increase or decrease the peak-to-peak displacement during the time T osc and / or by periodically changing the peak-to-peak of the oscillation as a sine wave, triangular wave or square wave, for example, during the time T osc can be executed with a peak-to-peak displacement that changes during the oscillation step.

[0018] In some embodiments of the method for manipulating a liquid, the vibration step can be performed by vibrating at least a portion of a wall of a capillary channel at a frequency that is at or approximately the resonant frequency ω of the wall of the capillary channel. The resonant frequency ω of the wall can vary, for example, as a function of the tension in the wall of the capillary channel and / or as a function of the composition and structure of the wall. For example, the vibration frequency can increase as the tension in the wall increases and decrease as the tension in the wall decreases. The resonant frequency ω of the wall can be determined by vibrating the wall at a frequency ω using an actuator, such as a piezoelectric actuator (e.g., a piezoelectric bender), and then stopping the vibration of the wall at the frequency ω. When the wall is no longer driven by the actuator, the wall under tension continues to move with a magnitude of motion related to the efficiency of the vibration driven by the actuator at the frequency ω. The magnitude of the motion can be determined, for example, by using a displacement transducer to convert the wall motion into an electrical signal. The displacement transducer can be an actuator used to vibrate the wall at a frequency ω, and its operating mode can be reversed from that of an actuator to that of a displacement transducer. After determining the magnitude of the wall's motion in response to the wall being vibrated at frequency ω, the system again uses the actuator to vibrate the wall, but at a different frequency ω. For example, the system can reverse the operation of the displacement transducer to act as an actuator again. The system then repeats the steps of ceasing to drive the wall to vibrate, determining the magnitude of the vibration, and vibrating the wall at a different frequency. The determined magnitude is greatest when the vibration frequency corresponds to the resonant frequency ω. Once the resonant frequency ω is determined, the system continues to drive the wall to vibrate at or substantially the same frequency as the resonant frequency ω. To ensure that the vibration remains at or near frequency ωr, the system may drive vibration at or near frequency ωr for several cycles, then stop driving the vibration of the wall at frequency ωr, determine the magnitude of the vibration, and vibrate the wall at a different frequency ωr'.Here, ωr’ is a frequency in the vicinity of the frequency ωr (for example, within about 3% - 10%). Depending on whether the determined magnitude of the wall vibration is greater or smaller than the vibration at the frequency ωr, the system may continue with the steps of stopping driving the wall vibration, determining the magnitude of the vibration, and vibrating the wall at different frequencies, and may maintain the vibration at the frequency that is the resonance frequency of the wall or a frequency substantially the same as it. For example, the steps of stopping the wall vibration (driving), determining, and (re)driving may be repeated at least once for every Nth vibration. Here, N is about 500 or less, about 250 or less, about 125 or less, or about 75 or less.

[0019] In some embodiments of the method of manipulating a liquid, the position of the liquid - gas interface with respect to the longitudinal axis of the capillary channel may remain substantially unchanged after N vibrations, where N can be, for example, at least about 500, at least about 1000, at least about 2000, or at least about 3000. The position of the liquid - gas interface with respect to the longitudinal axis of the capillary channel may remain substantially unchanged after N vibrations, where N can be, for example, about 20,000 or less, about 15,000 or less, about 10,000 or less, or about 5,000 or less. After said N vibrations, the position of the liquid - gas interface can be, for example, about 2 mm or less, about 1 mm or less, or about 750 μm or less of its initial position along the longitudinal axis of the capillary channel.

[0020] In some embodiments of the method of manipulating a liquid containing a cavity, the opening of each of the one or more cavities can be the essentially sole or the sole path for the gas to enter and exit the cavity. When the opening is the essentially sole path for the gas to enter and exit the cavity, the other paths are in total insufficient to prevent the formation of a second liquid - gas interface adjacent to the sidewall of the capillary channel. The vibration can be performed at a frequency the same as or substantially the same as the resonance frequency of the wall of the capillary channel, and the resonance frequency can vary, for example, as a function of the tension of the wall of the capillary channel and / or the composition and structure of the wall.

[0021] In some embodiments of the method of manipulating a liquid, a portion of the capillary channel may have a length L along the longitudinal axis of the capillary channel. In some embodiments including a cavity, the ratio of the total volume of a plurality of cavities disposed along a portion of the capillary channel having the length L to the total volume of the capillary channel along the length L excluding the cavities may be at least about 0.03, at least about 0.05, at least about 0.075, at least about 0.085, at least about 0.1, at least about 0.125, or at least about 0.15. The ratio of the total volume of a plurality of cavities disposed along a portion of the capillary channel having the length L to the total volume of the capillary channel along the length L excluding the cavities may be about 0.4 or less, about 0.3 or less, about 0.25 or less, about 0.225 or less, or about 0.2 or less. The ratio of the total area of the openings of a plurality of cavities disposed along a portion of the capillary channel having the length L to the total inner surface area of the capillary channel along the length L excluding the area occupied by the openings of the cavities may be at least about 0.0075, at least about 0.009, at least about 0.011, at least about 0.012, or at least about 0.013. The ratio of the total area of the openings of a plurality of cavities disposed along a portion of the capillary channel having the length L to the total inner surface area of the capillary channel along the length L excluding the area occupied by the openings of the cavities may be about 0.05 or less, about 0.04 or less, about 0.03 or less, about 0.02 or less, about 0.0175 or less, or about 0.015 or less.

[0022] In some embodiments of the method of manipulating a liquid, the manipulation may further include inducing a bulk movement of the liquid along the longitudinal axis of the capillary channel after and / or while simultaneously vibrating the pressure of the gas. For example, the liquid-gas interface may induce a bulk movement of the liquid along, for example, the capillary channel for a total time T movDuring this time, it can be moved from a first position within the capillary channel to a second position that is a distance D away from the first position along the longitudinal axis of the capillary channel. The first position may be distal or proximal to the second position along the longitudinal axis of the capillary channel. Time T mov is, for example, at least about 1 second, at least about 2 seconds, at least about 3 seconds, or at least about 4 seconds. Time T mov is, for example, about 12.5 seconds or less, about 10 seconds or less, or about 7.5 seconds or less. The step of moving the liquid-gas interface is performed over time T mov and can be carried out by increasing or decreasing the gas pressure of the gas adjacent to the liquid during this time. When the gas pressure is increased, bulk movement of the liquid is induced in a first direction along the longitudinal axis of the capillary channel, and when the gas pressure is decreased, bulk movement of the liquid is induced in a second, opposite direction. The movement of the liquid in response to the changing gas pressure tends to counteract the change, and as a result, the gas pressure is essentially the same at the start and end of time T mov The step of increasing or decreasing the gas pressure can be carried out by increasing or decreasing the compression of the walls of the capillary channel. For example, increasing or decreasing the compression can be, respectively, at the end of time T mov by an amount of at least about 7.5 μm, at least about 12.5 μm, at least about 17.5 μm, or at least about 22.5 μm, decreasing or increasing the internal width of the capillary channel along an axis substantially perpendicular to its longitudinal axis compared to its width at the start. The step of oscillating the gas pressure is carried out during at least a portion of time T mov at least a portion of time T mov substantially all of time T mov essentially all of time T mov or the entire time T.

[0023] In some embodiments of the method of manipulating a liquid, the length L of the portion of the capillary channel can be, for example, at least about 0.5 mm, at least about 1 mm, at least about 2 mm, at least about 3 mm, or at least about 4 mm. The length L can be, for example, about 25 mm or less, about 17.5 mm or less, about 10 mm or less, about 7.5 mm or less, about 6 mm or less, or about 5 mm or less. The length L can be an N-fold multiple of the distance along the longitudinal axis of the capillary channel between the proximal wall of the first cavity and the proximal wall of the cavity disposed distally adjacent thereto. N can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6. N can be, for example, about 25 or less, about 20 or less, about 15 or less, about 12 or less, about 10 or less, about 8 or less, or about 6 or less. The distance D can independently have any of the same dimensions as the length L.

[0024] In some embodiments of the method of manipulating a liquid, the capillary channel can be a microchannel, such as an analysis channel, within a microfluidic channel network of a microfluidic device (such as a microfluidic strip). The wall of the microchannel is a layer of the microfluidic strip, such as a substrate.

[0025] In some embodiments of the method of manipulating a liquid, the vibration step can be performed by vibrating an actuator that contacts the outer surface of the wall of the capillary channel. The actuator can be a piezoelectric actuator, such as a piezoelectric bender.

[0026] In some embodiments, a method includes introducing a sample liquid into a microchannel of a microfluidic device (e.g., a microfluidic strip), the sample liquid occupying a first portion of the microchannel, a second portion of the microchannel adjacent to the first portion being occupied by a gas, the sample liquid and the gas forming a liquid-gas interface therebetween. The method then includes repeatedly imparting energy to the gas in the second portion of the microchannel. At least a portion of the energy is transferred from the gas to the sample liquid through the liquid-gas interface.

[0027] In some embodiments, a method of applying energy to a liquid disposed within a capillary channel and having a plurality of liquid-gas interfaces includes applying energy to the liquid at a frequency substantially similar to the resonant frequency of the liquid with respect to the liquid-gas interfaces. The method may include inducing a bulk movement of the liquid along the longitudinal axis of the capillary channel following and / or while simultaneously applying energy to the liquid. The capillary channel may include a plurality of openings disposed along its sidewalls, and the liquid may contact gas at each of the one or more openings, and one of the plurality of liquid-gas interfaces may be adjacent to, for example, the sidewall of the capillary channel there. Each of the plurality of liquid-gas interfaces may have an axis of symmetry (axis of symmetry) at an angle other than zero with respect to the axis of symmetry of the longitudinal axis of the capillary channel at the position of such liquid-gas interface. The non-zero angle may be at least about 20°, at least about 35°, at least about 45°, at least about 67.5°, or at least about 90°. The non-zero angle may be about 160° or less, about 145° or less, about 130° or less, or about 120° or less. For example, the axis of symmetry of the liquid-gas interface and the longitudinal axis of the capillary channel may be substantially perpendicular to each other. Each of the openings may be an opening to a cavity containing at least one gas of the liquid-gas interfaces. Each of the one or more cavities may have a longitudinal axis having an angle of at least about 20°, at least about 35°, at least about 45°, at least about 67.5°, or at least about 85°, with respect to the longitudinal axis of the capillary channel at the position of the opening of the cavity to the capillary channel. Each of the one or more cavities of the capillary channel may have a longitudinal axis having an angle of about 160° or less, about 145° or less, about 135° or less, or about 120° or less, with respect to the longitudinal axis of the capillary channel at the position of the opening of the cavity to the capillary channel. For example, the longitudinal axis of each of the plurality of cavities and the longitudinal axis of the capillary channel at the position of the opening of such cavity to the capillary channel may be substantially perpendicular to each other.

[0028] In some embodiments of the method, energy is imparted to the liquid and such cavities are included, where the net effect of imparting energy is configured and arranged such that it induces little or no force to propel the liquid along the longitudinal axis of the capillary channel. For example, upon energy impartation, the net effect of a plurality of side cavities disposed within the reagent or detection zone of the capillary channel mobilizes the dry reagent present therein, mixes the sample liquid with the reagent disposed therein, and / or incubates the reaction between the target and the reagent disposed therein, and may induce only a force insufficient to propel the liquid out of such a reagent or detection zone for a sufficient time. In some embodiments, the longitudinal axis of each of the first set of cavities is oriented at a first angle with respect to the longitudinal axis of the capillary channel, and the longitudinal axis of each of the second set of cavities is oriented at a second angle with respect to the longitudinal axis of the capillary channel, and the first and second angles are opposite (opposing) each other. For example, the opening of each of the first set of cavities may face substantially proximally within the capillary channel, and the opening of each of the second set of cavities may face substantially distally within the capillary channel. Alternatively, or in combination, the longitudinal axis of each of the plurality of cavities and the longitudinal axis of the capillary channel at the location of such a cavity within the capillary channel, for example within the reagent or detection zone of the capillary channel, may be substantially perpendicular to each other.

[0029] In some embodiments of the method, energy is imparted to the liquid and such cavities are included, and in each of one or more cavities, the opening may be the essentially sole or the sole path for gas to enter and exit the cavity. When the opening is the essentially sole path for gas to enter and exit the cavity, other paths are, in total, insufficient to prevent the formation of a second liquid-gas interface adjacent to the sidewall of the capillary channel. The vibration can be carried out at a frequency that is the same as or substantially the same as the resonance frequency of the walls of the capillary channel, and the resonance frequency can vary, for example, as a function of the tension of the walls of the capillary channel and / or the composition and structure of the walls.

[0030] In some embodiments of the method of imparting energy to the liquid, the step of imparting energy can be carried out by repeatedly changing the pressure of the gas adjacent to the liquid-gas interface of the liquid. The step of repeatedly increasing and decreasing the pressure of the gas can be carried out by vibrating the walls of the microchannel. Here, the walls are in direct communication with the gas, for example, directly above or below the gas, but are not in direct communication with the liquid, for example, not directly above or below the liquid. For example, a portion of the wall that is vibrated can be disposed at least about 0.2 cm, at least about 0.3 cm, at least about 0.5 cm, at least about 0.75 cm, at least about 1.00 cm, at least about 1.25 cm, or at least about 1.5 cm away from the liquid-gas interface of the liquid along the longitudinal axis of the capillary channel.

[0031] In some embodiments of the method of imparting energy to a liquid, the step of imparting energy may be performed at an acoustic frequency, for example, about 15,000 Hz or less, about 10,000 Hz or less, about 5,000 Hz or less, about 3,000 Hz or less, about 2,000 Hz or less, about 1,750 Hz or less, about 1,500 Hz or less, about 1,250 Hz or less, about 1,150 Hz or less, about 1,050 Hz or less, or about 950 Hz or less. The vibration step may be performed at about 25 Hz or more, about 50 Hz or more, about 100 Hz or more, about 150 Hz or more, about 200 Hz or more, about 250 Hz or more, about 500 Hz or more, about 750 Hz or more, or about 900 Hz or more.

[0032] In some embodiments, a microfluidic device (e.g., a microfluidic strip) includes a microfluidic channel network and first and second conductive leads. A first portion of each conductive lead is disposed within a respective different liquid sensing location of the microfluidic channel network. Each liquid sensing location is a location of the microfluidic device that is occupied by liquid during use of the microfluidic device. A second portion of each conductive lead is disposed at a different mechanical sensing location of the microfluidic device. Each mechanical sensing location is a location within the microfluidic device where a mechanical operation and / or movement of the microfluidic device and / or about the microfluidic device changes the electrical characteristics of the respective conductive lead. In some embodiments, the microfluidic device includes a conductive bridge member configured to change the electrical characteristics of at least one (e.g., both) of the second portions of the first and second conductive leads during a mechanical operation and / or movement of the microfluidic device or about the microfluidic device. For example, the conductive bridge member may increase or decrease the impedance or resistance between the first and second portions during a mechanical operation and / or movement of the microfluidic device or about the microfluidic device. At least one (e.g., both) of the respective mechanical sensing locations may be a location configured to be maintained in a dry state, for example, not occupied by liquid, during use of the microfluidic device.

[0033] In some embodiments, a method of using a microfluidic device (e.g., a microfluidic strip) includes: (i) mechanically changing the shape and / or morphology of the microfluidic device to sense the occurrence and / or extent of the mechanical change by detecting a first electrical signal at at least one of a first electrical contact and a second electrical contact of the microfluidic device; (ii) detecting a second electrical signal at least at the first electrical contact to sense the presence of a liquid at at least one first location within a microfluidic channel network of the microfluidic device and / or to perform at least one electrochemical determination, e.g., regarding the presence and / or amount of a second target; and (iii) detecting a third electrical signal at least at the second electrical contact to sense the presence of a liquid at at least one second location within a microfluidic channel network of the microfluidic device and / or to perform at least one electrochemical determination, e.g., regarding the presence and / or amount of a second target, wherein the at least one second location is spaced apart from the at least one first location within the microfluidic channel network of the microfluidic device. In some embodiments, the third electrical signal results from a change in impedance, e.g., a change in continuity, between respective portions of first and second conductive leads, each of the first and second conductive leads being in electrical communication with one of the first and second electrical contacts, respectively. The step of sensing the presence of a liquid at the at least one first location may include sensing an electrical signal resulting from a first electrode in contact with a sample liquid at the at least one first location, the first electrode being in electrical communication with a first conductive lead and the first contact, and the step of sensing the presence of a liquid at the at least one second location may include sensing an electrical signal resulting from a second electrode in contact with a sample liquid at the at least one second location, the second electrode being in electrical communication with a second conductive lead and the second contact.

[0034] In some embodiments, a method of changing the volume of a gas bladder of a microfluidic device (e.g., a microfluidic strip) comprises providing a microfluidic device including a microfluidic channel network, a gas bladder in gas communication with the microfluidic channel network, and a gas bladder sensor in perceptible communication with the gas bladder. An actuator may be used to change (e.g., decrease) the volume of the gas bladder to release gas from the gas bladder into the microfluidic channel network and / or to change (e.g., increase) the volume of the gas bladder to draw gas from the microfluidic channel network into the gas bladder. The release of gas from the gas bladder moves liquid present in the microfluidic channel network in a first direction therein, and the drawing of gas into the gas bladder moves such liquid in a second different (e.g., opposite) direction therein. The method comprises changing (e.g., decreasing and / or increasing) the volume of the gas bladder to a first extent using an actuator, sensing at least one gas bladder signal from the gas bladder sensor indicative of the first extent of the volume change and at least one actuator signal indicative of the extent of actuation corresponding to the first extent of the volume change, and storing at least the actuator signal or a signal indicative thereof. After the step of changing the volume to the first extent, the method comprises further changing (e.g., decreasing and / or increasing) the volume of the gas bladder by moving liquid in the microfluidic channel at least once using an actuator. After the step of moving the liquid, the method comprises changing the volume of the gas bladder to a second extent having a predetermined relationship to the first extent, as determined from the stored actuator signal or a signal indicative thereof, using an actuator.

[0035] In some embodiments of the method of changing the volume of the gas bladder of the microfluidic device, a first degree of volume change may correspond to the state in which the gas bladder is fully compressed operationally. A second degree of volume change may be substantially the same as the first degree of volume change, for example, may be essentially the same as the first degree of volume change.

[0036] In some embodiments of the method of changing the volume of the gas bladder of the microfluidic device, the gas bladder sensor includes either embodiment of the first and second conductive leads. For example, the gas bladder sensor may include the first and second electrode leads and a bridge contact configured to bring the first and second leads into an electrically conductive state when the gas bladder is changed to the first degree. The first and second electrode leads may each be in electrical communication with respective electrodes configured to sense the presence of liquid within the microfluidic channel network.

[0037] In some embodiments of the method of changing the volume of the gas bladder of the microfluidic device, the actuator is an actuator of a reader configured to operate the microfluidic device to determine the presence or amount of one or more targets in the sample liquid and / or to determine the physiological characteristics of the sample liquid. The actuator may be a piezoelectrically driven actuator. The actuator may be able to compress the outer wall of the gas bladder and reduce its volume.

[0038] In some embodiments, the microfluidic channel network includes first and second electrodes. Each of them has respective portions disposed within the microfluidic channel network at respective different positions so as to be in contact with at least the liquid present within the microfluidic channel network. When a liquid (e.g., a reagent liquid such as a sample liquid or a buffer solution) is disposed within the microfluidic channel network and connects the first and second electrodes (e.g., extends therebetween), it reduces the impedance or resistance between the first and second electrodes as compared to a gas such as air. Thereby, an electrical signal applied to the first electrode can be detected at the second electrode in the presence of the liquid. However, if one or more portions of the microfluidic channel network disposed between the first and second electrodes are not completely occupied by the sample liquid, but are occupied by a gas such as air, the electrical signal is not detected at the second electrode.

[0039] In some embodiments of a microfluidic channel network including first and second electrodes, the microfluidic channel network may include a plurality of interconnected microchannels. The first and second electrodes may be disposed within the same or different microchannels of the microchannel network. In some embodiments, the shortest distance between the first and second electrodes along one or more microchannels of the microchannel network is at least about 1 cm, at least about 1.5 cm, at least about 2 cm, or at least about 2.5 cm. In some embodiments, the microfluidic channel network includes one or more additional second electrodes at which an electrical signal can be detected in the presence of a sample liquid, and each of such additional second electrodes is disposed at a different position within the microfluidic channel network.

[0040] In some embodiments of a microfluidic channel network including a first and a second electrode, the microfluidic channel network can be formed within a microfluidic device (e.g., a microfluidic strip). The electrodes can be connected via conductive leads to a portion of the strip away from the microchannel network, e.g., at the periphery of the strip. An electrical signal can be introduced to the first electrode by the conductive lead and detected at the second electrode and one or more additional electrodes.

[0041] A method of using any of the embodiments of a microfluidic channel network including a first and a second electrode includes generating an electrical signal at the first electrode and determining whether the electrical signal is present at the second electrode. The electrical signal can be a time-varying signal such as a sine wave, a square wave, or a triangular wave. The time-varying signal can have a DC offset, and the DC offset can be of a sufficient magnitude such that the time-varying signal is substantially single-polarity (e.g., positive or negative) with respect to the ground potential, e.g., essentially single-polarity or completely single-polarity.

[0042] In some embodiments, the method includes providing a microfluidic device having a microchannel network that includes a first and a second electrode and two or more channels (e.g., analysis channels). The first electrode is in electrical communication with a first location within the microchannel network that is disposed away from each of the two or more channels. The second electrode is in electrical communication with the microchannel network at a second location disposed away from the first location and the two or more channels, at a third location disposed within the first channel, and at a fourth location disposed within the second channel. A sample liquid applied to the strip establishes continuity between the first and second electrodes along each of three paths within the microchannel network. That is, (1) between the first and second locations along a path excluding the first and second channels, (2) between the first location and the third location within the first channel, and (3) between the first location and the fourth location within the second channel. A time-varying signal can be applied (impressed) to the first electrode at the first location, for example, by applying the time-varying signal to a contact (junction) of the first electrode that can be positioned at or near the periphery of the strip. The time-varying signal received by the second electrode at the second, third, and / or fourth locations can be measured, for example, at a contact (junction) of the second electrode that can be positioned at or near the periphery of the strip. Based on the received signal, a reader can determine whether the liquid is filling the microchannel network between the first location and the second, third, and / or fourth locations, or combinations thereof.

[0043] In some embodiments, a method of moving a liquid includes moving the liquid in a first direction along a capillary channel and detecting a first electrical signal indicating that the liquid has contacted a first electrode disposed within the capillary channel. After detecting the first electrode signal, the method includes stopping the movement of the liquid and then moving the liquid in a second, opposite direction along the capillary channel. When starting or after starting the movement of the liquid in the second direction, the method may include detecting an end of the first electrode signal indicating that the liquid has moved away from the first electrode, e.g., that the liquid is no longer in contact with the first electrode. The method may include detecting a second electrical signal indicating that the liquid has contacted a second electrode disposed within the capillary channel and has moved away from the first electrode in the second direction. Detection of the second electrical signal may be performed during at least a portion of the time of performing the step of moving the liquid in the second direction. The method may include detecting an end of the second electrode signal indicating that the liquid has moved away from the second electrode, e.g., that the liquid is no longer in contact with the second electrode. After detecting the end of the second electrode signal, the method may include stopping the movement of the liquid in the second direction.

[0044] In some embodiments of the method of moving a liquid, the first electrical signal indicating that the liquid has contacted a first electrode disposed within the capillary channel may indicate that the liquid-gas interface of the liquid has displaced gas (vapor) from the position of the first electrode as the liquid moves in the first direction. The end of the first electrode signal may indicate that gas (vapor) has again occupied the position of the first electrode as the liquid-gas interface moves in the second direction. The end of the second electrode signal may indicate that gas has occupied the position of the second electrode and that the liquid-gas interface of the liquid has advanced in the second direction away from the first electrode and passed the second electrode.

[0045] In some embodiments of the method of moving a liquid, after stopping the movement of the liquid in a second direction, the method includes repeating the steps of moving the liquid in a first direction, detecting a first electrical signal, and stopping moving the liquid in the first direction. After repeating the step of stopping moving the liquid in the first direction, the method may include repeating the steps of moving the liquid in a second opposite direction, detecting a second electrical signal, detecting the stop of the second electrical signal, and stopping the movement of the liquid in the second direction. The series of steps may be repeated N times. Here, N is at least 2, at least about 5, at least about 10, at least about 20, or at least about 25.

[0046] In some embodiments of the method of moving a liquid, the first and second electrodes are arranged at a distance D apart along the capillary channel, and the distance D is, for example, at least about 0.5 mm, at least about 1 mm, at least about 2 mm, at least about 3 mm, or at least about 4 mm. The distance D can be, for example, about 25 mm or less, about 17.5 mm or less, about 10 mm or less, about 7.5 mm or less, about 6 mm or less, or about 5 mm or less. The movement of the liquid in the first or second direction can be performed at a speed of at least about 0.2 mm / s, at least about 0.5 mm / s, at least about 0.75 mm / s, or at least about 1.0 mm / s. The movement of the liquid in the first or second direction can be performed at a speed of about 4 mm / s or less, about 3 mm / s or less, about 2 mm / s or less, or about 1.5 mm / s.

[0047] In some embodiments of the method of moving a liquid, one or both of the first and second electrodes are disposed adjacent to at least a first hydrophobic layer, or at least first and second hydrophobic layers, disposed within the capillary channel. Each of the hydrophobic layers may cover a first portion of the electrode within the capillary channel. For example, the first and second hydrophobic layers may cover respective first portions of the electrodes. The covered first portions of the electrodes may be disposed adjacent to opposing sidewalls of the capillary channel, leaving a second uncovered portion of the electrode disposed in a central portion of the capillary channel along an axis transverse to the longitudinal axis of the capillary channel.

[0048] In some embodiments of the method of moving a liquid, the method includes vibrating the gas pressure of the gas of the liquid-gas interface while moving the liquid in a first direction and / or a second direction. The liquid-gas interface may be a first liquid-gas interface having an axis of symmetry generally aligned with the longitudinal axis of the capillary channel. The capillary channel may include one or more openings disposed along its sidewall, and the liquid contacts the gas at each of the one or more openings to form a second liquid-gas interface there. In some embodiments of the method of moving a liquid, each of the one or more second liquid-gas interfaces may have an axis of symmetry generally perpendicular to the axis of symmetry of the first liquid-gas interface and the longitudinal axis of the capillary channel. The vibration may be performed at a frequency that is the same as or substantially the same as the resonance frequency of the liquid within the capillary channel that communicates with the second liquid-gas interface.

[0049] In some embodiments of the method of moving a liquid, each of the one or more openings disposed in the sidewall may be an opening to a cavity containing the gas of the second liquid-gas interface. The opening of each of the one or more cavities may be the only path for the gas to enter and exit the cavity.

[0050] In some embodiments of the method of moving a liquid, the capillary channel can be a capillary channel within a microfluidic channel network of a microfluidic strip. The first and second electrodes can be connected via conductive leads to a part of the strip remote from the microchannel network, for example at the periphery of the strip. The first and second electrical signals can be detected by the conductive leads.

[0051] In some embodiments, a microfluidic device (e.g., a microfluidic strip) contains a reagent. The microfluidic device can include a first and a second substantially flat layer, such as a substrate, each having an opposing surface. The opposing surfaces of the first and second layers are spaced apart and disposed, for example, by at least one third layer that fixes, e.g., adheres, the first and second layers in an opposing state. The at least one third layer occupies a region less than all of the region between the first and second layers, and the microfluidic channel network is at least partially defined by the unoccupied portion of the region between the first and second layers. The inner opposing surfaces of the first and second layers not occupied by the third layer define the respective upper and lower inner surfaces of the microchannel network, and the respective inner surfaces of the third layer adjacent to the unoccupied portion of the region between the first and second layers define the sidewalls of the microchannel network. The reagent is disposed on at least one opposing surface of the first and second layers within one channel of the microfluidic channel network. At least a first portion of the reagent is disposed within the channel on a portion of such an opposing surface not occupied by at least a portion of the at least one third layer. At least a second portion of the reagent is disposed outside the channel on a portion of such an opposing surface occupied by the at least one third layer. The third layer is located above the second portion of the reagent. The second portion of the reagent can be disposed adjacent to, e.g., abutting, the first portion of the reagent outside the first sidewall of the channel. At least a third portion of the reagent is disposed outside the channel on a portion of such an opposing surface occupied by the at least one third layer outside the second sidewall of the channel of the microfluidic channel network. The second sidewall faces the first sidewall across the channel. The third layer is located above the second portion of the reagent.

[0052] In some embodiments of a microfluidic device containing a reagent, the third layer may define a plurality of cavities adjacent to the microchannel network. The capillary channel may include one or more openings disposed along its sidewalls, and the liquid contacts the gas at each of the one or more openings, forming a second liquid-gas interface there. Each of the one or more second liquid-gas interfaces may have an axis of symmetry that is generally perpendicular to the axis of symmetry of the first liquid-gas interface and the longitudinal axis of the capillary channel.

[0053] In some embodiments, a method of manufacturing a microfluidic device (e.g., a microfluidic strip) comprises providing a first and a second layer (e.g., a substrate), depositing a reagent on a portion but not all of a first surface of the first layer, disposing a first surface of at least one third layer on the first surface of the first layer, and disposing a first surface of the second layer on a second surface of the at least one third layer, wherein (i) the at least one third layer occupies an area smaller than the areas of the first surface of the first layer and the first surface of the second layer, and at least a first portion of the third layer is on top of at least a portion but not all of the deposited reagent, and the first and second layers are fixed relative to each other so as to face each other, and (ii) at least a portion of the first surface of the first layer not occupied by the third layer and at least a portion of the first surface of the second layer not occupied by the third layer define first and second inner surfaces of a microfluidic channel network, and at least a portion of the deposited reagent is deposited on the first surface of the first layer within the microfluidic channel network.

[0054] In some embodiments of the method of manufacturing a microfluidic device, the method may include depositing a reagent deposition boundary on a first surface of a first substrate prior to the step of depositing the reagent. The reagent deposition boundary defines the extent of the area occupied by the reagent upon deposition on the first surface of the first substrate. The reagent deposition boundary may be formed of a hydrophobic layer or film, such as an ink. At least a portion (e.g., most, essentially all, or all) of the reagent deposition boundary may be deposited on a portion of the first surface of the first layer and overlaid by a third layer.

[0055] In some embodiments of the method of manufacturing a microfluidic device, the method may include providing a plurality of side cavities within an edge of a third layer adjacent to a portion of the first surface of the first and second layers where the reagent is deposited and not occupied by the third layer. In use, each cavity forms a liquid-liquid interface with the liquid present within the microfluidic channel network.

[0056] In some embodiments of the method of manufacturing a microfluidic device, the microfluidic strip (e.g., device) may be configured to perform an assay for determining the presence and / or amount of at least one target present in a liquid applied to the microfluidic device.

[0057] In some embodiments, a microfluidic device (e.g., a microfluidic strip) includes a microfluidic channel network that includes a sample application zone, a common branch channel in fluid communication with the sample application zone, and a plurality of analysis channels. Each of the analysis channels has a proximal origin connected to the common branch channel at a first position along it and a distal end spaced from the proximal origin by such an analysis channel. Each of the first positions may be different from the other first positions. The microfluidic channel network includes a vent in gas communication with the common branch channel. For each of the plurality of analysis channels, the proximal origin provides the only path through which liquid and gas can enter and exit such an analysis channel. Each analysis channel includes, for example, a gas bladder adjacent to or defining its distal end. Compressing the gas bladder of an analysis channel reduces the volume of the gas bladder and releases gas from the gas bladder toward the proximal origin of such an analysis channel. When sample liquid is present within an analysis channel, it is moved along such an analysis channel in a direction from the gas bladder toward the proximal origin of such an analysis channel. Decompressing the gas bladder of an analysis channel increases the volume of the gas bladder and draws gas into the gas bladder from such an analysis channel. When sample liquid is present within an analysis channel, it is moved along such an analysis channel toward the decompressed gas bladder.

[0058] In some embodiments, the vent and the sample application zone are the only paths through which gas can enter and exit the microfluidic channel network. In some embodiments, the vent is spaced from the common branch channel by at least a vent channel. The vent channel may have a cross-sectional area of about 20,000 mm 2 or less, about 18,000 mm 2 or less, or about 17,000 mm 2 or less. The vent channel is at least about 5,000 mm 2 at least about 10,000 mm 2 or at least about 12,500 mm 2may have a cross-sectional area. The vent channel may have a length of at least about 7500 mm, at least about 10,000 mm, or at least about 12,500 mm. The vent channel may have a length of about 20,000 mm or less, or about 17,500 mm or less. In some embodiments, each of the analysis channels has a length of at least about 10,000 mm, at least about 15,000 mm, or at least about 17,500 mm. The analysis channel may have a length of about 35,000 mm or less, about 30,000 mm or less, or about 27,000 mm or less.

[0059] In some embodiments, the analysis channel is a plurality of first analysis channels, the microfluidic network includes a second analysis channel, the second analysis channel has a proximal origin connected to a common branch channel at a second position along it, and is gas-connected to a vent at its distal end. For example, the vent channel may include the distal end of the vent and the proximal origin connected to the distal end of the second analysis channel. The second analysis channel may be configured to determine (decide) the hematocrit of a blood sample applied to the sample application zone of the microfluidic device. The second position may be different from each of the first positions.

[0060] In some embodiments, the microfluidic device includes a distal portion configured to be received within a reader during operation of the microfluidic device. Each of the gas bladders is located within the distal portion of the microfluidic device. The microfluidic device includes a proximal portion configured to protrude from the reader during operation of the microfluidic device. The sample application zone and the vent are located within the proximal portion of the microfluidic device.

[0061] In some embodiments, a microfluidic device (e.g., a microfluidic strip) includes a microchannel network having a sample application port and a supply channel extending from the sample application zone. The microfluidic device includes at least one zone of soluble anticoagulant disposed within the sample application port, the supply channel, or a combination thereof. The soluble anticoagulant can be in a dry state. The at least one zone of soluble anticoagulant can be (i) disposed within or adjacent to the sample application port, or both, or (ii) within the supply channel and spaced from the sample application port. When within the supply channel and away from the sample application port, the at least one zone of soluble anticoagulant can be spaced from the sample application port by a length of the supply channel, e.g., by a length of at least about 3 mm, at least about 5 mm, at least about 7.5 mm, or at least about 10 mm. This length portion is essentially free or (entirely) free of soluble anticoagulant. The at least one zone of anticoagulant can be a first zone of anticoagulant disposed within or adjacent to the sample application port, and the microfluidic device can include a second zone of soluble anticoagulant (e.g., in a dry state) disposed within the supply channel and spaced from the first zone of anticoagulant by a length of the supply channel, e.g., by a length of at least about 3 mm, at least about 5 mm, at least about 7.5 mm, at least about 10 mm, at least about 12.5 mm, or at least about 15 mm. This length portion is essentially free or (entirely) free of soluble anticoagulant. The soluble anticoagulant can include or can be essentially composed of lithium heparin.

[0062] In some embodiments, the method comprises introducing a sample, such as a blood-based sample, into a sample application port of a microfluidic device, and flowing the sample along a microchannel extending from the sample application port within the microfluidic device. The flowing step includes contacting the sample with a first zone of an anticoagulant disposed within or adjacent to the sample application port, and a second zone of an anticoagulant disposed within the channel and separated from the sample application port and the first zone of the anticoagulant by a length of the channel that is essentially free (or entirely free) of soluble anticoagulant. The length can be, for example, at least about 3 mm, at least about 5 mm, at least about 7.5 mm, at least about 10 mm, at least about 12.5 mm, or at least about 15 mm. The soluble anticoagulant can be in a dry state prior to contacting the sample. The soluble anticoagulant can include lithium heparin or can consist essentially of lithium heparin. The method can further include combining (mixing) the sample contacted with the soluble anticoagulant with a reagent within a channel of the microfluidic device, and performing a diagnostic assay, such as an immunoassay, for the presence of one or more targets within the sample using the reagent. The one or more targets can be, for example, an antigen of a coronavirus such as SARS-CoV-2.

[0063] In some embodiments, a microfluidic device (e.g., a microfluidic strip) includes a microfluidic channel network that includes a plurality of microchannels. One or more of the microchannels include at least one first inner surface. Liquid within the one or more microchannels contacts the first inner surface. The inner surface is substantially diffusely reflective within at least one wavelength band. Within the wavelength band, at least 50%, at least 65%, at least 75%, at least 90%, at least 95%, or at least 99% of the light reflected from incident light hitting the surface at an angle between about 0° and about ±45° relative to the normal surface when the surface is dry is diffusely reflected rather than directly reflected at the angle of incidence. Within the wavelength band, the diffuse reflection can be, for example, substantially uniform, can be, for example, Lambertian, and can be preferential with respect to lobes or maxima of reflectivity in a particular direction. The reflectivity of the diffusely reflective surface can be at least 90%, at least 92%, at least 95%, or at least 97.5% within a 100 nm wide wavelength band in the range of 400 nm to 2500 nm, 600 nm to 2200 nm, or 800 nm to 1500 nm.

[0064] In some embodiments, the diffusely reflective surface includes a metal oxide such as aluminum oxide, or a crystalline material or mineral such as barium sulfate. The microfluidic device can include a layer, e.g., a polymer layer, and the diffusely reflective inner surface can be a coating or layer applied over at least a portion of the total area of the layer.

[0065] With respect to the longitudinal axis of the one or more microchannels, the diffusely reflecting inner surface can have a length of at least about 1 mm, at least about 2 mm, at least about 3 mm, or at least about 4 mm, and / or about 10 mm or less, about 7.5 mm or less, or about 6 mm or less. At the location of the diffusely reflecting inner surface, the microchannel can have a width along an axis perpendicular to the longitudinal axis of at least about 500 μm, at least about 750 μm, or at least about 1000 μm, and / or a width of about 2000 μm or less, about 1500 μm or less, or about 1250 μm or less. The diffusely reflecting inner surface can occupy substantially all of the width and / or area of the inner surface of the channel within the length of the diffusely reflecting inner surface.

[0066] In some embodiments, the microfluidic device (e.g., a microfluidic strip) is configured to perform a serological immunoassay (e.g., a bridging serological assay) for antibodies against SARS-CoV-2. The microfluidic strip includes a microfluidic channel network that includes a sample application port and an analysis channel in fluid communication therewith. The analysis channel includes a first reagent and a second reagent. The first reagent includes the SARS-CoV-2 spike glycoprotein S1 subunit, or a fragment thereof. The second reagent includes the SARS-CoV-2 receptor binding domain (RBD), or a fragment thereof. In certain embodiments, the first and second reagents include the SARS-CoV-2 S1 spike glycoprotein. When a fragment of the spike glycoprotein S1 subunit is used, the fragment retains the ability to specifically bind to an antibody against the SARS-CoV-2 spike glycoprotein S1 subunit, and such an antibody can be present in a mammal (e.g., a human) as a result of a previous or current infection with SARS-CoV-2. When a fragment of the SARS-CoV-2 RBD is used, the fragment retains the ability to specifically bind to an antibody against the SARS-CoV-2 RBD, and such an antibody can be present in a mammal (e.g., a human) as a result of a previous or current infection with SARS-CoV-2.

[0067] In some embodiments, one of the first reagent and the second reagent is bound to, or configured to bind to, a capture agent (e.g., a surface such as the surface of the channels of a microchannel network or a particle such as a magnetic particle), and the other of the first reagent and the second reagent is bound to, or configured to bind to, a detectable label. For example, the first reagent can be a conjugate comprising (i) the SARS-CoV-2 S1 spike glycoprotein S1 subunit or a fragment thereof, and (ii) a binder configured to bind to a capture agent (e.g., a surface such as the surface of the channels of a microchannel network or a particle such as a magnetic particle). For example, the conjugate can comprise one of biotin and streptavidin, and the particle or surface can comprise the other of biotin and streptavidin. For example, the first reagent can be a conjugate of the SARS-CoV-2 spike glycoprotein S1 subunit or a fragment thereof and biotin, and the microfluidic strip can further comprise particles conjugated (bound) to streptavidin, such as magnetic particles. The second reagent can be a conjugate comprising (i) the SARS-CoV-2 RBD or a fragment thereof, and (ii) a detectable label such as a fluorescent particle, e.g., a fluorescent latex particle, etc.

[0068] In some embodiments, a method of performing a serological immunoassay for antibodies against SARS-CoV-2 comprises combining (mixing) a liquid sample suspected of containing such antibodies, such as a blood-based sample, with a first reagent comprising the SARS-CoV-2 spike glycoprotein S1 subunit or a fragment thereof, and a second reagent comprising the SARS-CoV-2 RBD or a fragment thereof, and determining (deciding) the presence and / or amount of a complex comprising the first reagent, the antibody, and the second reagent. The method may include applying the liquid sample to a sample application zone of a microfluidic device that includes one or both of the first and second reagents in a microfluidic channel network of the microfluidic device. The first reagent may be a conjugate comprising (i) the SARS-CoV-2 spike glycoprotein S1 subunit or a fragment thereof and (ii) a binder configured to bind to particles such as surface or magnetic particles. For example, the first reagent may be a conjugate comprising (i) the SARS-CoV-2 spike glycoprotein S1 subunit or a fragment thereof and (ii) biotin, and the method may further include combining (mixing) the liquid sample with a third reagent comprising a conjugate of magnetic particles and streptavidin. In certain embodiments, the first reagent may be a conjugate comprising (i) the SARS-CoV-2 spike glycoprotein S1 subunit or a fragment thereof and (ii) biotin that binds to a conjugate comprising magnetic particles and streptavidin prior to introduction of the sample. The second reagent may be a conjugate comprising (i) the SARS-CoV-2 RBD or a fragment thereof and (ii) a detectable label such as fluorescent particles, such as fluorescent latex particles, etc. In some embodiments, the first, second, and / or third reagents are disposed within an analysis channel of the microfluidic channel network.The distal portion of the analysis channel may include a gas bladder, and the method may include, as disclosed herein, compressing, decompressing, and / or vibrating the gas bladder, and may manipulate the liquid sample, for example, move the liquid sample and / or mix the liquid sample with a reagent as disclosed herein. The method may include magnetically holding the complex of the third reagent, the first reagent, the antibody against SARS-CoV-2, and the second reagent within the detection zone of the microfluidic channel network prior to detecting the complex. The method may include discharging the sample liquid from the detection zone as disclosed herein prior to the detection step.

[0069] In some embodiments, a microfluidic device (e.g., a microfluidic strip) is configured to perform an assay for detecting an antigen, e.g., a SARS-CoV-2 antigen, in a sample, e.g., a nasal sample, a nasopharyngeal sample, or a saliva sample. The sample may be, for example, from a blood-based sample such as blood, plasma or serum, or from a nasal or nasopharyngeal swab specimen, and / or may be contained within a universal transport medium (UTM) or a viral transport medium (VTM). The sample may contain blood, serum or plasma. For example, the sample may have serum and / or plasma, or may consist essentially of serum and / or plasma. In certain embodiments, the sample may not be exposed to a lysis step (e.g., a lysis step sufficient to lyse white blood cells, red blood cells, or viruses such as coronaviruses such as SARS-CoV-2) prior to the detection assay. In certain embodiments, the step of exposing the sample to the binding assay is performed without releasing the coronavirus antigen from the cells present in the sample, e.g., without releasing the coronavirus antigen from white blood cells, red blood cells, or either white blood cells or red blood cells. In certain embodiments, the step of exposing the sample to the binding assay can be performed without first contacting the sample with a chemical lysis reagent, e.g., without first contacting the sample with an alkali, detergent or enzyme at a concentration sufficient to rupture the walls of the cells present in the sample, e.g., the walls of white blood cells, red blood cells, or either white blood cells or red blood cells. In certain embodiments, the step of exposing the sample to the binding assay can be performed without first exposing the sample to a physical lysis step, e.g., without first exposing the sample to thermal conditions, osmotic pressure, shear forces or cavitation sufficient to rupture the walls of the cells present in the sample, e.g., the walls of white blood cells, red blood cells, or either white blood cells or red blood cells.In certain embodiments, the step of exposing the sample to the binding assay is performed without first exposing the sample to a lysis step sufficient to lyse the coronavirus within the sample, e.g., without first exposing the sample to a lysis step sufficient to lyse SARS-CoV-2 present within the sample. In certain embodiments, when a coronavirus antigen is detected, substantially all of the detected coronavirus antigen is a free-form antigen, e.g., an antigen not associated with an intact virus.

[0070] In certain embodiments, the method includes a step of aggregating red blood cells in a volume of blood to prepare a sample. For example, the method may include contacting the volume of blood with an antibody against a protein produced by or associated with red blood cells, such as an antibody against glycophorin A, or an aggregating protein, such as phytohemagglutinin E. The aggregating step may be performed, for example, by introducing the volume of blood into a microfluidic device and contacting the blood in the channels of the microfluidic device with an antibody produced by or associated with red blood cells or an aggregating protein in the microfluidic device. In certain embodiments, the method includes a step of separating a sample of plasma and / or serum from red blood cells. In certain embodiments, the step of separating a sample of plasma and / or serum is performed without passing the plasma and / or serum through a filter. The step of separating a sample of plasma and / or serum may be performed within a portion of a microfluidic channel having a substantially smooth inner surface. For example, a portion of the microfluidic channel may not include protrusions having a height greater than about 10%, 7.5%, 5%, or about 2.5% of the width or height of the microfluidic channel, or may have an inner surface that does not include protrusions configured to delay the movement of red blood cells along the longitudinal axis of the microfluidic channel relative to the movement of plasma and / or serum along the longitudinal axis. In certain embodiments, the step of separating a sample of plasma and / or serum is performed within a portion of a microfluidic channel having at least one internal turn of at least about 90 degrees.

[0071] The microfluidic strip includes a microfluidic channel network that includes a sample application port and an analysis channel in fluid communication therewith. The analysis channel includes a first reagent and a second reagent. The first and second reagents include a binding agent, such as an antibody, that binds to a SARS-CoV-2 antigen. As used herein, unless otherwise specified, the term “antibody” is understood to mean an intact (full-length) antibody (e.g., an intact monoclonal antibody) or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), which is understood to include an intact (full-length) antibody, an antigen-binding fragment, or an Fc fragment that has been modified, engineered, or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies (bispecific antibodies). Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0072] In certain embodiments, the microfluidic device can include reagents for different assays in different microchannels within the same device (e.g., a microfluidic strip). For example, in certain embodiments, reagents for detecting anti-coronavirus antibodies can be present in one microchannel, and reagents for detecting coronavirus antigens can be present in another microchannel of the same device. In certain embodiments, reagents for detecting anti-coronavirus antibodies or coronavirus antigens can be present in one microchannel, and control reagents can be present in another microchannel of the same device.

[0073] In some embodiments, one of the first reagent and the second reagent is bound, or configured to bind, to a capture agent (e.g., a surface such as the surface of the channels of a microchannel network, or a particle such as a magnetic particle), and the other of the first reagent and the second reagent is bound, or configured to bind, to a detectable label. For example, the first reagent can be a conjugate comprising (i) a first antibody against a SARS-CoV-2 antigen (e.g., nucleocapsid), and (ii) a binder configured to bind to a particle such as a surface or a magnetic particle. For example, the conjugate can comprise one of biotin and avidin or streptavidin, and the particle or surface can comprise the other of biotin and avidin or streptavidin. For example, the first reagent can be a conjugate of (i) a first anti-SARS-CoV-2 nucleocapsid antibody, or a fragment thereof, and (ii) biotin, and the microfluidic strip can further comprise particles conjugated (bound) to streptavidin, such as magnetic particles. In another example, the conjugate can comprise (i) a first anti-SARS-CoV-2 nucleocapsid antibody and (ii) biotin, which is bound to a conjugate comprising magnetic particles and streptavidin prior to introduction of the sample. The second reagent can be a conjugate comprising (i) a second antibody against a SARS-CoV-2 antigen and (ii) a detectable label such as a fluorescent particle, e.g., a fluorescent latex particle, etc. In certain embodiments, the first SARS-CoV-2 antibody binds to a different epitope on the SARS-CoV-2 antigen than the second SARS-CoV-2 antibody binds to. In certain embodiments, the first reagent and / or the second reagent is bound to, or configured to bind to, a single capture agent or detectable label. In any of the above embodiments, the antibody can be a Fab.

[0074] In some embodiments, a method of performing an assay for detecting an antigen, such as a SARS-CoV-2 antigen, may include combining (mixing) a liquid sample, which may be contained within a universal transport medium (UTM) or a viral transport medium (VTM) and is suspected of containing such an antibody, such as a nasal sample, a nasopharyngeal sample, or a saliva sample, with a first reagent containing a first antibody against a SARS-CoV-2 antigen (e.g., nucleocapsid) and a second reagent containing a second antibody against a SARS-CoV-2 antigen (e.g., nucleocapsid), and determining the presence and / or amount of a complex containing the first reagent, the antigen, and the second reagent. The method may include applying the liquid sample to a sample application zone of a microfluidic device. In some embodiments, the sample has a volume between about 10 microliters and about 50 microliters. In some embodiments, the sample is not purified and / or concentrated before being applied to the sample application zone. The microfluidic device may contain one or both of the first and second reagents in the microfluidic channel network of the microfluidic device. The first reagent may be a conjugate containing (i) a first antibody against a SARS-CoV-2 antigen (e.g., nucleocapsid) and (ii) a binder configured to bind to a capture agent (e.g., a surface such as the surface of a channel in the microfluidic channel network or a particle such as a magnetic particle). For example, the first reagent may be a conjugate containing (i) a first SARS-CoV-2 anti-nucleocapsid antibody and (ii) biotin, and the method may further include combining (mixing) the liquid sample with a third reagent containing a conjugate of magnetic particles and streptavidin. In another embodiment, the first and third reagents may be bound before the introduction of the sample (e.g., bound before being dried in the microchannel). The second reagent may be a conjugate containing (i) a second SARS-CoV-2 anti-nucleocapsid antibody and (ii) a detectable label such as a fluorescent particle, e.g., a fluorescent latex particle, etc. In some embodiments, the first, second, and / or third reagents are disposed within an analysis channel of the microfluidic channel network.The distal portion of the analysis channel may include a gas bladder, and the method may include compressing, decompressing, and / or vibrating the gas bladder, as disclosed herein, and may manipulate the liquid sample, for example, move the liquid sample and / or mix the liquid sample with a reagent as disclosed herein. The method may include magnetically retaining the complex of the third reagent, the first reagent, the antibody against SARS-CoV-2, and the second reagent within the detection zone of the microfluidic channel network prior to detecting the complex. The method may include discharging the sample liquid from the detection zone as disclosed herein prior to the detection step.

[0075] In some embodiments, the sensitivity of the SARS-CoV-2 antigen assay is at least about 96%, at least about 97%, at least about 98%, or at least about 99% PPA (positive percent agreement) using a reference PCR test. In certain embodiments, the SARS-CoV-2 antigen assay can detect SARS-CoV-2 antigen in a sample when the virus is present in the sample in an amount sufficient for detection of viral nucleic acid at about 28 - 34 RT-PCR cycles, about 29 - 34 RT-PCR cycles, about 30 - 34 RT-PCR cycles, about 31 - 34 RT-PCR cycles, about 32 - 34 RT-PCR cycles, about 33 - 34 RT-PCR cycles, about 29 - 33 RT-PCR cycles, about 30 - 33 RT-PCR cycles, about 31 - 33 RT-PCR cycles, about 32 - 33 RT-PCR cycles, about 29 - 32 RT-PCR cycles, about 30 - 32 RT-PCR cycles, about 31 - 32 RT-PCR cycles, about 29 RT-PCR cycles, about 30 RT-PCR cycles, about 31 RT-PCR cycles, about 32 RT-PCR cycles, about 33 RT-PCR cycles, or about 34 RT-PCR cycles (i.e., "Ct" value). Exemplary PCR (e.g., RT-PCR) assays include, for example, the cobas® SARS-CoV Test (see Roche Diagnostics, www.fda.gov / media / 136049 / download), and the Abbott Real Time SARS-CoV Assay (see Abbott Molecular, www.molecular.abbott / sal / 9N77-095_SARS-CoV-2_US_EUA_Amp_PI.pdf).

[0076] In some embodiments, the sensitivity of the assay is at least about 96%, at least about 97%, at least about 98%, or at least about 99% PPA (positive percent agreement) when the sample is taken on the day of onset of symptoms, up to 1 day after onset of symptoms, up to 2 days after onset of symptoms, up to 3 days after onset of symptoms, up to 4 days after onset of symptoms, up to 5 days after onset of symptoms, up to 6 days after onset of symptoms, up to 7 days after onset of symptoms, up to 8 days after onset of symptoms, up to 9 days after onset of symptoms, up to 10 days after onset of symptoms, up to 11 days after onset of symptoms, or up to 12 days after onset of symptoms. In certain embodiments, the sensitivity of the assay is at least about 96%, at least about 97%, at least about 98%, or at least about 99% PPA (positive percent agreement) when the sample is taken between about 5 to about 12 days after onset of symptoms. In certain embodiments, the detection limit of the SARS-CoV-2 antigen assay is from about 25 - 35 TCID50 / ml, about 28 - 33 TCID50 / ml, about 30 - 33 TCID50 / ml, about 31 - 33 TCID50 / ml, about 31 - 32 TCID50 / ml, about 32 - 33 TCID50 / ml, or about 32 TCID50 / ml.

[0077] In some embodiments, a method of preparing a plasma sample comprises: (i) mixing (combining) a blood sample containing red blood cells with an agglutination reagent; and (ii) separating the mixed blood sample and agglutination reagent in a microchannel of a microfluidic device into a red blood cell portion disposed in a first portion of the microchannel and a plasma portion disposed in a second portion of the microchannel. The red blood cell portion contains substantially all, e.g., essentially all, of the red blood cells of the blood sample mixed with the agglutination reagent. The plasma portion consists essentially of the plasma of the blood sample mixed with the agglutination reagent, e.g., the plasma portion can consist essentially of the plasma of the blood sample. The mixing can be performed in a microchannel of a microfluidic device.

[0078] The blood sample can be a whole blood sample from a mammal, such as a human. The blood sample can be obtained, for example, from a venous draw or a finger stick. The plasma portion, which consists essentially of the plasma of the blood sample, is a plasma sample suitable for determining the presence and / or amount of one or more targets therein, for example, an immunological determination. Exemplary targets include C-reactive protein (CRP), D-dimer, troponin complex (troponin-T, troponin-I, or troponin-C, etc.), glucose, and lipids (cholesterol, HDL, or LDL, etc.). The number and amount of red blood cells that may remain in the plasma portion are sufficiently small and at a sufficiently low concentration so as not to substantially interfere with the use of the plasma portion as a plasma sample for such determination, if present.

[0079] The microfluidic device used in a method for preparing a plasma sample can include a liquid sample introduction port in fluid communication with a microchannel, and the microchannel can include an aggregation reagent disposed therein. The combining step can include introducing a blood sample into the microchannel via the liquid sample introduction port and flowing the blood sample along the microchannel to combine (mix) the blood sample with the aggregation reagent disposed therein.

[0080] The separation step in a method of preparing a plasma sample may include a step of sequentially arranging an erythrocyte portion and a plasma portion along the axis of the flow in a microchannel. The separation step in a method for preparing a plasma sample may include a step of forming a liquid-liquid interface between the erythrocyte portion and the plasma portion, wherein one liquid of the liquid-liquid interface is the liquid of the erythrocyte portion and the other liquid of the liquid-liquid interface is the plasma of the plasma portion. The (both) liquids of the liquid-liquid interface may be similar, for example, essentially identical, in composition and / or miscibility. For example, the liquid of the erythrocyte portion may include residual plasma surrounding the erythrocytes therein. Thus, in any embodiment, the liquid-liquid interface may be substantially defined by a substantial change in the local concentration of erythrocytes incorporated in the liquid (the erythrocyte concentration is significantly higher in the erythrocyte portion than in the plasma portion) rather than a substantial difference in the miscibility of the liquids of the two portions.

[0081] In some embodiments of a method for preparing a plasma sample, the method includes a step of forming a distal liquid-gas interface disposed within the microchannel and spaced from the ambient gas surrounding the microfluidic device by at least the erythrocyte portion and the plasma portion, wherein the liquid of the distal liquid-gas interface is one of the erythrocyte portion or the plasma portion. For example, in an embodiment where the microfluidic device includes a sample introduction port, the proximal portion of the blood sample, e.g., the proximal gas-liquid interface, may remain exposed to the ambient gas through the sample introduction port, while the distal liquid-gas interface is spaced from the sample introduction port and the ambient gas by at least the erythrocyte portion and the plasma portion present within the microchannel. As another example, the microchannel may include a vent in gas communication with the ambient gas, and the distal liquid-gas interface may be spaced from the vent and the ambient gas therein by at least the erythrocyte portion and the plasma portion present within the microchannel. The liquid of the distal liquid-gas interface may be the plasma of the plasma portion.

[0082] In some embodiments, a method for preparing a plasma sample includes the step of mixing (combining) the plasma of the plasma portion with one or more reagents disposed within a microchannel, wherein the one or more reagents are configured to interact with a target present within the plasma portion. The one or more reagents may include at least one reagent configured to participate in a reaction with the target to facilitate its determination (decision). For example, the one or more reagents may participate in a binding reaction with the target, such as an immunological reaction with the target, and may be, for example, an antibody or a fragment thereof configured to bind to the target. The method may further include the step of determining (deciding) the presence and / or amount of the target within the plasma portion based at least in part on the interaction of at least one reagent with the target. For example, the one or more reagents may include a detectable label, such as a fluorescent particle, such that the binding of the reagent to the target can be determined.

[0083] A method for preparing a plasma sample may include the step of maintaining a liquid-liquid interface during combining (mixing) the plasma of the plasma portion with one or more reagents disposed within a microchannel. The method may further include the step of maintaining the liquid-liquid interface during determining (deciding) the presence and / or amount of a target within the plasma portion. In some embodiments, the area of the liquid-liquid interface is essentially the same as the cross-sectional area of the microchannel at the position of the liquid-liquid interface within the microchannel and is defined by and is the same as the cross-sectional area, for example. For example, the area of the liquid-liquid interface may be at least about 0.03 mm 2 at least about 0.04 mm 2 at least about 0.06 mm 2 at least about 0.07 mm 2 or at least about 0.08 mm 2 and may be. Also, the area of the liquid-liquid interface may be about 0.25 mm 2 or less, about 0.2 mm 2 or less, about 0.175 mm 2 or less, about 0.15 mm 2 or less, about 0.135 mm 2Less than or about 0.12 mm 2 Less than or about 0.1 mm 2 It may be less than.

[0084] In a method of preparing a plasma sample, a method of separating a combined (mixed) blood sample and an agglutination reagent may include, for example, flowing the combined (mixed) blood sample and the agglutination reagent in a first direction along a microchannel and then flowing the mixture in a second direction opposite to the first direction along the microchannel to vibrate the combined (mixed) blood sample and the agglutination reagent. The step of separating the combined (mixed) blood sample and the agglutination reagent may include the step of flowing the mixture in the first direction and then the step of flowing the combined (mixed) blood sample and the agglutination reagent in the second direction, and repeating the steps at least N times, where N is, for example, at least about 3, at least about 5, at least about 7, or at least about 10, and may be, for example, about 20 or less, about 15 or less, or about 10 or less. The step of flowing the mixture in the first direction and / or the second direction may include moving the distal liquid-gas interface of the combined (mixed) blood sample and the agglutination reagent through a volume in the microchannel of at least about 0.1 μL, at least about 0.25 μL, at least about 0.35 μL, at least about 0.45 μL, or at least about 0.55 μL. The step of flowing the mixture in the first direction and / or the second direction may include moving the distal liquid-gas interface of the combined (mixed) blood sample and the agglutination reagent through a volume in the microchannel of about 2 μL or less, about 1.5 μL or less, about 1.2 μL or less, about 1 μL or less, about 0.9 μL or less, about 0.8 μL or less, or about 0.7 μL or less. The step of flowing the mixture in the first direction and / or the second direction may include moving the distal liquid-gas interface of the combined (mixed) blood sample and the agglutination reagent through a length along the microchannel of at least about 1 mm, at least about 2 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm.The step of flowing the mixture in the first direction and / or the second direction may include moving the distal liquid-gas interface of the combined (mixed) blood sample and the aggregation reagent along a length along a microchannel of about 10 mm, about 7.5 mm, about 6.5 mm, or about 5.5 mm. The step of flowing in the first direction may be performed by either increasing or decreasing the pressure of the gas at the distal liquid-gas interface of the combined (mixed) blood sample and the aggregation reagent, and the step of flowing in the second direction may be performed by the other of increasing or decreasing the pressure of the gas at the distal liquid-gas interface.

[0085] Any method of preparing a plasma sample can be carried out without passing the plasma portion through a filter, such as a membrane. The method of preparing a plasma sample can be carried out without subjecting the blood sample to a deterministic lateral displacement sufficient to separate the blood sample into a red blood cell portion and a plasma portion, e.g., without subjecting the blood sample to any (at all) deterministic lateral displacement. The deterministic lateral displacement approach separates particles, such as red blood cells, from particles that are differentially displaced when forced around microstructures that impede the flow by flowing the particle-containing sample through an array of microstructures or micropillars.

[0086] In any method of preparing a plasma sample, the inner surface of a portion of the microchannel in which the separation step is performed may substantially lack protrusions or microstructures sufficient to preferentially retain red blood cells in an amount sufficient to separate the red blood cell portion from the plasma portion. The inner surface of a portion of the microchannel in which the separation step is performed may be impermeable and may lack pores through which liquid can flow by capillary action or other means.

[0087] In any method of preparing a plasma sample, the separation step can be carried out without subjecting the blood sample to an inertial focusing sufficient to separate the blood sample into a red blood cell portion and a plasma portion, e.g., without subjecting the blood sample to any substantial inertial focusing.

[0088] In any method of preparing a plasma sample, the separation step can be carried out without subjecting the blood sample to a centrifugal force sufficient to separate the blood sample into a red blood cell portion and a plasma portion, for example, without subjecting the blood sample to any substantial centrifugal force. The separation step can be carried out without rotating the microfluidic device. The separation step can be carried out without flowing the blood sample along a curved flow path in the microchannel.

[0089] In any method of preparing a plasma sample, the separation step can be carried out using a flow axis of a microchannel that is oriented substantially perpendicular to the local gravitational field of the Earth, for example, within about 20 degrees, within about 15 degrees, within about 10 degrees, or within about 5 degrees of perpendicular or substantially perpendicular to the local gravitational field of the Earth.

[0090] In any method of preparing a plasma sample, the volume of the plasma portion separated from the blood sample can be at least about 0.075 μL, at least about 0.1 μL, at least about 0.15 μL, at least about 0.175 μL, or at least about 0.2 μL. The volume of the plasma portion separated from the blood sample can be about 0.75 μL or less, about 0.65 μL or less, about 0.55 μL or less, about 0.45 μL or less, about 0.4 μL or less, about 0.35 μL or less, or about 0.325 μL or less.

[0091] In any method of preparing a plasma sample, the method can be carried out without combining (mixing) the blood sample with an anticoagulant, such as heparin or EDTA. The plasma portion may be essentially free of, for example, (entirely free of) an anticoagulant such as heparin or EDTA.

[0092] In any of the embodiments that include a reagent, the reagent can be selected from the group consisting of a lysis reagent, a buffer reagent, a reagent detectably labeled (e.g., a fluorescently labeled reagent), a reagent configured to specifically bind to a target to be detected, a magnetically labeled (tagged) reagent, or a combination thereof.

[0093] In any of the embodiments, the movement of a liquid, and / or mixing, and / or the vibration of the pressure of a gas at the liquid-gas interface of a liquid can be brought about by using an actuator such as a piezoelectric actuator like a piezoelectric bender to compress, decompress, and / or vibrate the walls of a microfluidic device or a capillary channel.

[0094] In any of the embodiments that include or use a microfluidic device (e.g., a strip), the microfluidic device can include a plurality of capillary channels, such as analysis channels. Each of the capillary channels (e.g., analysis channels) can have its own wall, such as the wall of a gas bladder, and each wall can be operable independently of the walls of other capillary channels of the microfluidic device, such as the wall of a gas bladder, and can allow independent control over the manipulation (e.g., mixing by vibration and / or flow) of a sample liquid within the corresponding analysis channel. A reader can be configured with a plurality of actuators, each of which can be configured to independently control the volume and / or vibration of the corresponding gas bladder. Each of the actuators can be configured to determine (decide) the respective resonance frequency ωr of the corresponding capillary channel and gas bladder and to vibrate the wall of the capillary channel at the frequency ωr, as described in the foregoing embodiments. One or more actuators of different gas bladders can be vibrated out of phase (e.g., in antiphase) with respect to one or more other actuators.

[0095] Whether referred to as a channel, microchannel, or capillary channel, such conduits are preferably sized and configured to allow a sample liquid to flow along them by capillary action. For example, the maximum dimension of such a conduit can be, along at least one, at least two, or any (number of) axes oriented perpendicular to the longitudinal axis of the conduit, in the portion intended to receive a liquid such as the sample liquid, about 2 mm or less, about 1.5 mm or less, about 1 mm or less, about 0.9 mm or less, about 0.75 mm or less, about 0.5 mm or less, about 0.25 mm or less, about 0.125 mm or less, or combinations thereof.

[0096] The terms "layer" and "substrate" are used synonymously herein. A layer of a microfluidic device, such as a substrate, can itself be composed of more than one layer, for example, along an axis substantially perpendicular to the plane of the microfluidic device. For example, the substrate of a microfluidic strip can be fixedly opposed (e.g., adhered) by a central layer composed of more than one layer, and for example, the central layer can include a central layer (of itself) and first and second adhesive layers to which the respective outer substrates are fixed. The walls of a microfluidic channel network can be defined by the absence, e.g., removal, of a portion of the central layer. A layer of a microfluidic device, such as a substrate, can itself be composed of more than one layer, for example, along an axis substantially parallel to the plane of the microfluidic device. For example, the substrate of a microfluidic strip can be fixedly opposed (e.g., adhered) by a central layer composed of a plurality of, e.g., spaced-apart, first and second separate layers, and their edges can at least partially define the walls of a microfluidic channel therebetween. A microfluidic device can include one or more layers (e.g., substrates), and each layer can itself be formed from one or more layers fixed together, separate layers spaced apart from each other, or combinations thereof.

[0097] One or more of the layers in any of the embodiments of a microfluidic device, such as a microfluidic strip, may be formed from a polymer such as polyester, polydimethylsiloxane (PDMS) elastomer, a thermoplastic resin, and combinations thereof. The microfluidic strip may be formed from a non-polymeric material, or may be formed from layers of different materials including, for example, one or more rigid layers formed from a polymer, quartz or silicon, and one or more flexible layers formed from, for example, a polymer. The adhesive layer in any of the embodiments of a microfluidic device, such as a microfluidic strip, may include one or more adhesive layers, and such layers may include, for example, an acrylic adhesive.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0127] Referring to FIG. 1, diagnostic system 101 includes a diagnostic reader 111 and a microfluidic strip 10. The reader 111 activates the strip 10 to determine the presence and / or amount of at least one target (e.g., a biomolecule such as a protein) present in a sample liquid applied to the strip 10. The reader 111 also activates the strip 10 to determine the physicochemical properties (e.g., hematocrit) of the sample liquid applied to the strip 10. The reader 111 includes an input port 113 for receiving the microfluidic strip 10 and a touch screen 115 by which a user can input and receive information related to the operation of the reader 111 and the determination of the target. The elements of the strip 10 are described first, followed by the elements of the reader 111.

[0128] Referring to FIGS. 2A and 2B, the strip 10 includes an upper substrate 12 and a lower substrate 14 each made of a 100 μm thick polyester film. The lower surface 12a of the upper substrate 12 and the upper surface 14a of the lower substrate 14 are adhered so as to face each other with a thickness of 110 μm of the adhesive layer 16. The adhesive layer 16 occupies an area smaller than the entire area of the surfaces 12a, 14a between the upper and lower substrates 12, 14 in order to define the microfluidic channel network 18. The microfluidic channel network 18 has a sample application zone 20, a common supply channel 22, a branch channel 24, an analysis channel 26, and a hematocrit channel 28. The microfluidic channel network 18 has side walls 30 defined by the adhesive layer 16, an upper wall 32 defined by a portion of the upper substrate 12 that is not occupied by the adhesive layer 16 (e.g., located above the non-occupied portion of the adhesive layer 16), and a lower wall 34 defined by a portion of the lower substrate 14 that is not occupied by the adhesive layer 16 (e.g., located below the non-occupied portion of the adhesive layer 16). The upper wall 32 has an inner surface 12a' defined by a portion of the surface 12a that is not occupied by the adhesive layer 16, e.g., exposed by the non-occupied portion of the adhesive layer 16. The lower wall 34 has an inner surface 14a' defined by a portion of the surface 14a that is not occupied by the adhesive layer 16, e.g., exposed by the non-occupied portion of the adhesive layer 16. The upper substrate 12 has an outer (upper) surface 12b, and the lower substrate 14 has an outer (lower) surface 14b.

[0129] The sample application zone 20 is a port 36 that extends through the upper substrate 12 and the adhesive layer 16 above the microfluidic strip 10 and defines the proximal origin of the microfluidic channel network 18. The port 36 places a plurality of channels of the microfluidic channel network 18 in gas communication with the gas (e.g., air) 38 of the ambient atmosphere. The sample liquid (e.g., blood) applied to the sample application zone 20 via the port 36 flows by capillary action along the common supply channel 22 to the branch channel 24, and along the branch channel 24, a first portion of the sample liquid flows by capillary action to the analysis channel 26 and a second portion of the sample liquid flows by capillary action to the hematocrit channel 28.

[0130] The hematocrit channel 28 is configured and arranged to facilitate reagent-free optical determination of the hematocrit of a liquid sample of blood applied to the sample application zone 20. In the direction proceeding distally from the branch channel 24, the hematocrit channel 28 includes a supply electrode 70, a hematocrit filling electrode 72, a hematocrit detection zone 74, and a vent 76. The portions of the hematocrit channel 28 disposed proximal and distal to the hematocrit detection zone 74 each have a height of 110 μm and a width of 670 μm. The hematocrit detection zone 74 has a height of 110 μm, a width of 2300 μm, and a length of 3 mm. The operation of hematocrit determination will be further described below.

[0131] The analysis channel 26 is configured and arranged to facilitate determination of the presence and / or amount of a target present in the sample liquid. Along the longitudinal axis a1 of the analysis channel 26 and proceeding distally from the branch channel 24, the analysis channel 26 includes a vent 40, a capillary stop 42, a first reagent zone 44, a plurality of side cavities 46, a first filling electrode 48, a second reagent zone 50, a second filling electrode 52, a detection zone 54, a third filling electrode 56, an interval channel 58, and a gas bladder 60.

[0132] The common supply channel 22, the branch channel 24, the first reagent zone 44, the second reagent zone 50, and the spacer channel 58 each have a height of 110 μm and a width of 670 μm. The first reagent zone 44 and the second reagent zone 50 each have a length of 4.4 mm and a volume of approximately 324 nL. The detection zone 54 has a height of 110 μm, a width of 1500 μm, a length of 5.4 mm, and a volume of approximately 890 nL. The spacer flow path 58 has a length of 1 mm. The total volume of the analysis channel 26 between the capillary stop 42 and the third filling electrode 56 is approximately 1.6 μL. The gas bladder 60 has a height of 110 μm, a width of 5.5 mm, a length of 11.4 mm, and a volume of approximately 6.9 μL. The aforementioned dimensions of the portion of the analysis channel 26, for example the width, are those excluding the side cavity 46, which is further described below.

[0133] The first reagent zone 44 contains the lysis reagent 62 deposited on the lower surface 14a'. The lysis reagent 62 is configured to lyse cells present in the sample liquid and release the target present in the intracellular material. The second reagent zone 50 contains the labeled binding reagent 64 deposited on the lower surface 14a'. The labeled binding reagent 64 includes a first portion (e.g., an antibody) that specifically binds to the target and a second portion that is a detectable fluorescent label. The binding of the target and the labeled binding reagent 64 forms a first complex. The detection zone 54 contains the magnetic binding reagent 66 deposited on the lower surface 14a'. The magnetic binding reagent 66 has a first portion (e.g., an antibody) that binds to the first complex and a second portion that is a magnetic particle. The binding of the first complex and the magnetic binding reagent 66 forms a second complex.

[0134] Each of the reagents 62, 64, 66 is in a dry form (e.g., lyophilized form). When the manufacture of the strip 10 is completed (e.g., after the deposited reagents 62, 64, 66 are dried within the microfluidic channel network 18 and the upper and lower substrates 12, 14 are fixed to each other, e.g., adhered by the adhesive layer 16), the strip 10 is free of liquid (e.g., the strip 10 does not contain a stored liquid reagent such as a buffer). During use, the only liquid applied to the strip 10 is the sample liquid containing the target to be determined. The strip 10 is configured not to require (e.g., is not configured to permit) the introduction of liquids other than the sample liquid containing the target to be determined.

[0135] As described above, with further reference to FIG. 3, the analysis channel 26 includes a plurality of side cavities 46 disposed within the sidewalls 30 of the first reagent zone 44, the second reagent zone 50, and the detection zone 54. The side cavities 46 include sidewalls 30a defined by portions where the adhesive layer 16 is absent, e.g., portions removed from between the upper and lower substrates 12, 14, and upper and lower walls (not shown) respectively defined by respective portions of the surfaces 12a, 14a of the upper and lower substrates 12, 14 located above and below the portion where the adhesive layer 16 is absent. Each side cavity 46 has a height of 110 μm, a width of 75 μm along the longitudinal axis al of the analysis channel 26, a depth of 700 μm along an axis a2 oriented perpendicular to the longitudinal axis a1, and a volume of 5.8 nL. The side cavities 46 are spaced apart from each other by a distance of 700 μm along the longitudinal axis al of the analysis channel 26. Each side cavity 46 has a single opening 68 facing the analysis channel 26, and the opening 68 faces the opening 68 of the side cavity 46 disposed within the opposite sidewall 30 of the analysis channel 26.

[0136] Referring to FIG. 3, the length L oriented substantially along the axis a1 extends from the proximal wall 46' of the first side cavity 46 to the proximal wall 46" of the adjacent distal cavity 46. Inside each of the first and second reagent zones 44, 50, along a portion of the capillary channel having the length L, the ratio of the total volume (2×5.8 nL) of the side cavity 46 to the total volume (57 nL) of the analysis channel 26 excluding the volume of the side cavity 46 is 0.20. Inside the detection zone 54, along the length corresponding to and oriented along the length L along the axis al, the ratio of the total volume (2×5.8 nL) of the side cavity 46 to the total volume (128 nL) of the analysis channel 26 excluding the side cavity 46 is 0.09. Inside each of the first and second reagent zones 44, 50, along the length L, the ratio of the total inner surface area (2×77,000 μm 2 +2×519,250 μm 2 ) of the analysis channel 26 excluding the opening 68 to the total surface area (2×8250 μm 2 ) of the opening 68 of the side cavity 46 is 0.0138. Inside the detection zone 54, along the length corresponding to and oriented along the length L along the axis al, the ratio of the total inner surface area (2×77,000 μm 2 +2×1,162,500 μm 2 ) of the analysis channel 26 excluding the opening 68 to the total surface area (2×8250 μm 2 ) of the opening 68 of the side cavity 46 is 0.0067.

[0137] In addition to the opening 68, the side cavities 46 lack means for the entry and exit of gas (vapor) and liquid and are sealed against the channel network 18 and the ambient atmosphere 38 elsewhere. The sample liquid 92 passing along the analysis channel 26 is prevented from fully entering the side cavities 46 by surface tension and the gas pressure of the gas 94 within the side cavities 46. The gas pressure increases as the sample liquid begins to enter the side cavities 46. Thus, the sample liquid within the analysis channel 26 and the gas 94 within each side cavity 46 form a gas-liquid interface 96 adjacent to the analysis channel 26. Each of the gas-liquid interfaces 96 has an axis of symmetry substantially aligned with the axis a2. The interaction between the side cavities 46 and the sample liquid is further described below. In FIG. 3, the sample liquid 92 has dissolved the labeled binding reagent 64 disposed within the second reagent zone 50, and thus the labeled binding reagent 64 is not shown. FIG. 3 also illustrates the distal liquid-gas interface 98 formed by the sample liquid 92 and the gas 100 present within the portion of the analysis channel 26 disposed distally of the sample liquid 92. The distal liquid-gas interface 98 is the liquid-gas interface of the sample liquid within the analysis channel 26 disposed away from the sample application zone 20 by the sample liquid. The distal liquid-gas interface 98 has an axis of symmetry substantially aligned with the longitudinal axis al. As described below, the position of the distal liquid-gas interface 98 changes as the determination of the target proceeds.

[0138] The gas bladder 60 defines the distal end of the analysis channel 26. The portion of the upper wall 32 above the gas bladder 60 defines the gas bladder upper wall 78, and the portion of the lower wall 34 below the gas bladder 60 defines the gas bladder lower wall 84. The gas bladder 60 is in gas communication with the ambient atmosphere 38 only via (i) the analysis channel vent 40 after passing through the analysis channel 26, (ii) the hematocrit channel vent 76 after passing through the analysis channel 26, the branch channel 24, and the proximal portion of the hematocrit channel 28, and (iii) the port 36 after passing through the analysis channel 26, the branch channel 24, and the common supply channel 22. When the manufacture of the strip 10 is complete, the strip 10 is typically packaged in an airtight sealed package, such as a foil pouch. If the strip 10 is opened in preparation for use, the gas within the microfluidic channel network 18 is free to exchange with the gas of the ambient atmosphere 36.

[0139] In addition to the vents 40, 76 and the port 36 described above, the microfluidic channel network 18 lacks ports or paths for the ingress and egress of gas (air) to the ambient atmosphere 38 and is sealed (airtight) against the ambient atmosphere 38 elsewhere. The microfluidic channel network 18 also lacks ports or other paths through which gas can be introduced into or withdrawn from the microfluidic network 18 via an external gas source outside the microfluidic strip 10. Thus, in the absence of sample liquid disposed within the microfluidic channel network 18 between the gas bladder 60 and the port 36, an increase in pressure within the gas bladder 60 (e.g., generated by reducing the volume of the gas bladder by compressing the upper wall 78 of the gas bladder toward the lower wall 84 of the gas bladder) vents the gas disposed proximally along the analysis channel 26, the branch channel 24, and the common supply channel 22 toward and out of the port 36 and, to a lesser extent, out of the vents 40, 76. In the absence of sample liquid disposed within the microfluidic channel network 18 between the gas bladder 60 and the port 36, a decrease in pressure within the gas bladder 60 (e.g., generated by increasing the volume of the gas bladder by retracting (withdrawing) the upper wall 78 of the gas bladder away from the lower wall 84 of the gas bladder) draws gas distally from the ambient atmosphere 38, through the port 36 and, to a lesser extent, through the vents 40, 76, into the microfluidic network 18 toward and into the gas bladder 60. Since the cross-sectional areas of the vents 40, 76 are significantly smaller than the cross-sectional area of the port 36, the primary path for the ingress and egress of gas to and from the microfluidic channel network during compression / expansion of the gas bladder 60 is the path through the port 36.

[0140] As described above with reference to FIG. 3 and as will be described further below, the sample liquid 92 disposed within the microfluidic channel network 18 between the port 36 and the gas bladder 60 forms a liquid-gas interface 98 that is the distal end of the sample liquid 92 and is located proximal to the gas bladder 60. Compression and retraction (withdrawal) of the upper wall 78 of the gas bladder increase and decrease, respectively, the gas pressure acting on the liquid-gas interface, providing the ability to control the flow and / or mixing of the sample liquid within the microfluidic channel network 18.

[0141] The electrodes of the strip 10 are configured and arranged to allow the reader 111 to monitor (monitor) the proper filling of the strip 10 with the sample liquid, the proper movement of the sample liquid within the strip 10, and the operation (e.g., compression state) of the gas bladder 60. Each of the supply electrode 70 and the filling electrodes 48, 52, 56, 72 is disposed on the inner surface 14a' of the lower wall 34 at a position where the sample liquid within the microchannel network 18 contacts the electrode. Each of the filling electrodes 48, 52, 56 of the analysis channel is connected to the distal peripheral portion 102 of the strip 10 via respective lead wires 48a, 52a, 56a. The hematocrit channel supply electrode 70 and the filling electrode 72a are each connected to the distal peripheral portion 102 via respective lead wires 70a, 72a. When the strip 10 is fully inserted into the reader 111, the distal ends of the lead wires 48a, 52a, 56a, 70a, 72a engage corresponding contacts (not shown) within the reader 111. The engaging contacts allow the reader 111 to transmit and / or receive electrical signals to and / or from the supply electrode 70 and / or the filling electrodes 48, 52, 56, 72. Except as described further below, the corresponding lead wires 48a, 52a, 56a, 70a, 72a are disposed on a portion of the upper surface 14a of the lower substrate 14 that is outside the microfluidic channel network 18 and remains covered by the adhesive layer 16.

[0142] Referring to FIG. 2A, the portion of the lead wire 48a of the first filling electrode 48 and the portion of the lead wire 56a of the third filling electrode 56 pass along the inner surface 14a' of the lower wall 84 of the gas bladder, respectively defining the first and second intervening conductive lead electrodes 48a', 56a'. The conductive bridge contact 86 is disposed on the inner surface 12a' of the upper wall 78 of the gas bladder and is positioned above the lead electrodes 48a', 56a'. As will be described below, when the upper wall 78 of the gas bladder is fully compressed, the bridge contact 86 establishes continuity between the lead electrodes 48a', 56a'. They are not directly continuous with each other in any other way. The reader 111 transmits and / or receives electrical signals to and from the lead electrodes 48a', 56a' via the same contact for the filling electrodes 48, 56.

[0143] The reader 111 and the strip 10 are configured to allow the reader 111 to determine when the strip 10 is fully inserted into the reader 111. For example, the reader 111 and the strip 10 may incorporate any of the exemplary structures and techniques for determining proper insertion of a strip into a reader, as disclosed in International Application PCT / GB2017 / 051946 (the "946 application"), filed on June 30, 2017. The application is hereby incorporated by reference in its entirety.

[0144] The reader 111 includes a magnetic field generator (not shown) for controlling the movement and / or positioning of the magnetic binding reagent 66. The magnetic field generator may incorporate any of the exemplary structures and techniques for magnetically controlling the movement and / or position of a magnetic reagent, as disclosed in the international application PCT / GB2019 / 053207 filed on November 12, 2019. This application is hereby incorporated by reference in its entirety. The magnetic field generator includes a permanent magnet at the end of a pivot arm and is configured to move the permanent magnet between a first position and a second position. In the first position, the magnet is separated from the detection zone 54 so that the detection zone 54 does not receive (experience) a magnetic field sufficient to substantially affect the magnetic particles of the magnetic binding reagent 66 within the detection zone 54. In the second position, the magnet is positioned below the lower substrate 14 below the detection zone 54 so that the magnetic particles of the magnetic binding reagent 66 receive (experience) a magnetic field that forces the magnetic particles towards the lower surface 35 of the lower substrate 14 within the detection zone 54. The forced movement force is sufficient to substantially hold the magnetic binding reagent 66 within the detection zone 54 in the presence of a flow and / or mixing of the sample liquid induced by a flow controller (as described hereinafter). With the strip inserted and the liquid sample not yet applied, the reader 111 positions the magnetic field generator in the first position.

[0145] The reader 111 comprises an optical detection system (not shown) having a light source configured to irradiate the detection zone 54 with light of a wavelength selected to excite fluorescence from a detectable label (tag) of the labeled binding reagent 64, and an optical detector configured to detect the fluorescence emitted therefrom. The optical detection system may include any of the exemplary structures and techniques for optical detection, as disclosed in the aforementioned "946 application".

[0146] To facilitate determination of the hematocrit, the reader 111 includes two light-emitting diodes (LEDs) (not shown), one of which emits light in the cyan region (506 nm) and the other of which emits light in the infrared region (805 nm). When the strip 10 is fully inserted into the reader 111, the LEDs are positioned above the hematocrit detection zone 74 and are configured to transmit light through the blood sample disposed therein. The diagnostic reader also includes a photodiode (not shown) configured to detect the light transmitted through the hematocrit detection zone 74. Hemoglobin strongly absorbs light in the cyan region (506 nm), while infrared light at 805 nm is not strongly absorbed by hemoglobin and thus allows for calibration of scatter and turbidity within the sample. The short optical path length (110 μm), determined by the height of the hematocrit detection zone, allows for the absorbance of hemoglobin to be measured in undiluted whole blood.

[0147] The reader 111 also includes a flow controller disposed therein. Referring to FIGS. 4 and 5, the flow controller includes an actuator such as a piezoelectric bender 117, which is an arm (like) extending from a fixed end 119 to an operating end 121. The piezoelectric bender 117 has a length of 30 mm along axis a1 and a width of 5 mm along axis a2 (defined below) (axes a1 and a2 are shown in FIG. 2A). The fixed end 119 is fixed to the mounting block 123 and is electrically coupled to the electrical connection portion 125, and the reader 111 provides an electrical actuation signal to the bender 117 through the electrical connection portion 125. The operating end 121 responds to an electrical signal, and the electrical signal controls the position and movement of the operating end 121 along an axis a3 (perpendicular to axes a1 and a2) oriented substantially perpendicular to the plane of the microfluidic strip 10. Then, the position and movement of the operating end 121 control the position and movement of the operating foot 127 along axis a3.

[0148] The actuating leg 127 is attached within the attachment block 123 below the actuating end 121 via the attachment pin 137 of the attachment block 123 passing through the slot 135 within the actuating leg 127. This attachment allows the actuating leg 127 to move freely along the axis a3 with respect to the attachment block 123. The actuating leg 127 has, along the axis a3, an upper surface 131, a lower surface 133, and an overall height of 8 mm between them. The upper surface 131 is located below the lower surface 129 of the actuating end 121 of the piezoelectric bender 117. The lower surface 133 is configured to transmit the movement of the actuating end 121 to the gas bladder upper wall 78 of the strip 10. When the strip 10 is fully inserted into the reader 111, the lower surface 133 of the actuating leg 127 contacts the contact portion 88 of the outer surface 12b of the gas bladder upper wall 78. The contact portion 88 has a length of 5 mm (along the axis a1 which is approximately aligned with the lengths of the analysis channel 26 and the gas bladder 60) and a width of 1 mm (along the axis a2 which is approximately perpendicular to the axis a1 and the lengths of the analysis channel 26 and the gas bladder 60). The area of the contact portion 88 is approximately 8% of the total area of the outer surface 12b of the gas bladder upper wall 78 over the gas bladder 60. The outer surface 14b of the lower substrate 14 of the strip 10 is placed on a strip support (not shown) within the reader 111. The strip support prevents the lower substrate 14 including the lower wall 34 from deflecting downward along the axis a3 in response to the downward movement of the actuating leg 127 (i.e., the movement towards the strip 10 along the axis a3) that compresses the gas bladder upper wall 78 as described below.

[0149] The contact portion 88 is laterally spaced from the third filling electrode 56 and is disposed distally therefrom along the axis a1. Accordingly, the contact portion 88 is disposed laterally spaced from the position of the analysis channel 26 occupied by the sample liquid during the operation of the microfluidic strip 10. For example, when the sample liquid occupies the first reagent zone 44 as determined by the first filling electrode 48 but has not yet progressed further distally along the analysis channel 26, the distance along the axis a1 between the distal liquid-gas interface 98 and the proximal portion 90 of the contact portion 88 is about 15 mm. When the sample liquid occupies the second reagent zone 50 as determined by the third filling electrode 56 but has not yet progressed further distally along the analysis channel 26, the distance along the axis a1 between the distal liquid-gas interface 98 and the proximal portion 90 of the contact portion 88 is about 10 mm. When the sample liquid occupies the detection zone 60 as determined by the hematocrit filling electrode 72, the sample liquid is at the most distal position within the analysis channel 26, and the distance along the axis a1 between the distal liquid-gas interface 98 and the proximal portion 90 of the contact portion 88 is about 5 mm.

[0150] When the reader 111 senses that the strip 10 is fully inserted and before the application of the sample liquid to the port 36, the reader 111 activates the flow controller to cause the piezoelectric bender 117 to press the lower surface 129 of the actuating end 121 against the upper surface 131 of the actuating foot 127. The applied pressure drives the actuating foot 127 downward along the axis a3, causing the lower surface 133 of the actuating foot 127 to compress the upper wall 78 of the gas bladder toward the lower wall 84 of the gas bladder. This compression places the upper wall 78 of the gas bladder under tension, causing the outer surface 12b of the upper wall 78 of the gas bladder to be substantially concave and the inner surface 12a' of the upper wall 78 of the gas bladder to be substantially convex. The upper substrate 12 including the upper wall 78 of the gas bladder is flexible (flexible) enough to allow compression and relaxation of the upper wall 78 over a distance corresponding to the height of the gas bladder 60.

[0151] The flow controller continues to compress the upper wall 78 of the gas bladder until the bridge contact 86 on the inner upper surface 12a' of the upper wall 78 of the gas bladder contacts the lead electrodes 48a', 56a' on the inner lower surface 14a' of the lower wall 84 of the gas bladder, bringing the lead electrodes 48a', 56a' into an electrically conductive state. The reader 111 receives a signal indicating that the upper wall portion 78 above the gas bladder 60 is fully compressed via the lead wires 48a, 56a in which the lead electrodes 48a', 56a' are in a conductive state. Next, the flow controller reverses the operation of the piezoelectric bender, retracts the operating end 121 in the vertical direction, and reduces the compression of the upper wall 78 of the gas bladder. Since the upper wall 78 of the gas bladder is under tension, the reduced compression causes the upper wall 78 of the gas bladder to retreat vertically with respect to the lower surface 133 of the operating foot 127, push the operating foot 127 vertically along the axis a3, separate the bridge contact 86 from the lead electrodes 48a', 56a', and interrupt the conduction with the lead wires 48a, 56a. The piezoelectric actuator continues to reduce the compression of the upper wall portion 78 until the signal of the lead wires 48a, 56a indicates that the conduction between the lead electrodes 48a', 56a' is interrupted. When a signal indicating the interruption of conduction is received, the piezoelectric actuator stops the further movement of the operating end 121, and maintains the compression of the upper wall portion 78 above the gas bladder 60 in a state where the bridge contact 86 and the lead electrodes 48a', 56a' are just separated (for example, by about 2.5 μm). At this time, the gas bladder 60 is in an operatively fully compressed state, the upper wall portion 78 is substantially concave and under tension, the contact portion 88 is pressed against the lower surface 133 of the operating foot 127, the upper surface 131 of the operating foot 127 is pressed against the lower surface 129 of the operating end 121, and the bridge contact 86 and the lead electrodes 48a', 56a' are only slightly separated.

[0152] The step of allowing the upper wall portion 78 to retreat slightly (recede) from the portion of the lower substrate 14 therebelow, thereby providing a slight separation between the bridge contact 86 (disposed on the inner surface 12a' of the upper wall portion 78) and the lead electrodes 48a', 56a' (disposed on the opposing inner surface 14a' of the lower substrate 14) provides several functions. For example, as will be described later, the first filling electrode 48 operates to sense the presence of sample liquid at the distal end of the first reagent zone 44, and the third filling electrode 56 operates to sense the presence of sample liquid at the distal end of the detection zone 54. If the bridge contact 86 maintains the electrical continuity between the lead electrodes 48a', 56a' (and thus the conductivity between the lead wires 48a, 56a and the filling electrodes 48, 56), the filling electrodes 48, 56 will not function to independently sense the presence of sample liquid. Interruption of the conduction between the lead electrodes 48a', 56a' allows the filling electrodes 48, 56 to perform their respective sample liquid sensing functions. Thus, a pair of lead wires (48a, 56a) allows for the performance of two separate (independent) liquid sensing functions (e.g., determining the presence of sample liquid at two respective positions via the electrodes 48, 56) and a mechanical sensing function (e.g., determining the compression of the gas bladder via the lead electrodes 48a', 56a'). Similarly, the reader 111 requires only a pair of contacts to engage with the lead wires 48a, 56a and receive the corresponding electrical signals indicative of sample liquid sensing and mechanical sensing. Thus, the strip 10 and the reader 111 are less expensive and less complex to manufacture than if separate pairs of independent electrodes and lead wires were used to sense the compression state of the gas bladder 60.

[0153] Also, during compression of the upper wall portion 78, the reader 111 receives from the piezoelectric actuator a calibration signal indicating the degree of compression required to fully compress the upper wall portion 78 and position the gas bladder 60 in an operatively fully compressed state. The reader 111 also receives a calibration signal indicating the amount of force required to be applied by the piezoelectric actuator to displace the upper wall portion 78 of the gas bladder 60. The reader 111 stores these calibration signals and can thus operate the piezoelectric actuator, without further signals from the lead electrodes 48a', 56a', to return the gas bladder 60 to an operatively fully compressed state and / or to achieve a given displacement of the upper wall portion 78. Such an ability is advantageous because sample liquid introduced subsequent to the analysis channel 26 during operation of the strip 10 (as described below) can render the electrodes 48, 56 conductive and render the lead electrodes 48a', 56a' inoperative or unreliable when sensing the compressed state of the gas bladder 60.

[0154] The retraction (recession) of the upper wall portion 78 also ensures that the upper wall portion 78 moves (e.g., for inflation or for further compression) without a delay in response to the movement of the actuating foot 127. Since the inflation and compression of the upper substrate 78 are used (as described later) to control the movement and / or mixing of the sample liquid in the analysis channel 26, the movement of the upper substrate 78 without a delay ensures that the controlled movement and / or mixing of the sample liquid occurs without a delay in response to the actuation by the piezoelectric actuator. If the step of slightly (precisely) separating the upper wall portion 78 from the portion of the lower substrate 14 thereunder is not carried out, an uncertain amount of retraction (recession) of the actuating foot 127 will occur before the occurrence of such separation and the start of the movement of the upper wall portion 78 (accompanied by a resulting volume change of the gas bladder 60). And the occurrence of a change (e.g., a stable change or an impulse) in the gas pressure in the gas bladder 60 that brings about the movement or mixing of the sample liquid in the analysis channel 26 will also be delayed. The absence of a delay means that the response of the upper wall portion 78 is substantially limited only by the physical properties (e.g., its elastic modulus) of the upper wall portion 78 and the mechanism of the actuating foot 27, and it rather means that there is no need to return the excessive compression of the upper wall portion 78 against the lower substrate 14 thereunder that may occur during the initial compression step.

[0155] After the step of positioning the gas bladder 60 in an operatively fully compressed state, the sample application zone 20 (port 36) maintains a state of gas communication with the surrounding ambient atmosphere 38, and when there is no sample liquid occupying the microchannel network 18, the gas bladder 60 and the remainder of the microchannel network 18 are in gas communication with the gas of the ambient atmosphere 38 surrounding the reader 111 and the microfluidic strip 10 and have the same gas pressure as that gas. The volume of gas displaced from the gas bladder 60 by placing the gas bladder 60 in an operatively fully compressed state as compared to the fully relaxed state is approximately the same as the volume of the analysis channel 26 between the branch channel 24 and the third filling electrode 56.

[0156] If the description of target determination continues, when the strip 10 is fully inserted into the input port 113 of the reader 111, the magnetic field generator is in the first position, and the gas bladder 60 is fully compressed operatively, the operator applies a sample liquid (e.g., blood) to the sample application zone 20 of the strip 10. The total volume of the applied sample is between 2.5 and 7.5 μL. The sample liquid flows through the port 36 and flows by capillary action until it reaches the branch channel 24 along the common supply channel 22. In the branch channel 24, the sample liquid branches into a first portion that travels along the branch channel 24 towards the hematocrit channel 28 and a second portion that travels along the branch channel 24 towards the analysis channel 26. The first portion of the sample liquid travels into the hematocrit channel 28 until the corresponding distal liquid-gas interface of the sample liquid (e.g., the liquid-gas interface of the sample liquid in the hematocrit channel 28 located away from the sample application zone 20 by the divided amount of the sample liquid in the hematocrit channel 28, the branch channel 24, and the common supply channel 22) just passes through the hematocrit channel vent 76. A small portion of the hematocrit channel 28 located distal to the vent 76 does not provide any path for the entry and exit of gas, so the gas pressure accumulated distal to the sample liquid stops the sample liquid from flowing along the hematocrit channel 28. The second portion of the sample liquid travels until the distal liquid-gas interface of the sample liquid (e.g., located away from the sample application zone 20 by the divided amount of the sample liquid in the analysis channel 26, the branch channel 24, and the common supply channel 22) just passes through the analysis channel vent 40 and contacts the capillary stop 42. At the capillary stop 42, the sample liquid stops flowing along the analysis channel 26. Due to the liquid-gas interface of the sample liquid at the positions defined in the above two paragraphs, the strip 10 is properly filled with the sample liquid and is ready to continue determining the presence and / or amount of the target present in the sample liquid.

[0157] The reader 111 is configured to determine the occurrence (or non-occurrence) of proper filling of the strip 10 with the sample liquid, as well as the presence of the sample liquid at positions within the microfluidic channel network 18 corresponding to the filling electrodes 48, 52, 56, 72. When the strip 10 is fully inserted into the reader 111, the reader 111 applies an electrical “supply” signal (e.g., a time-varying signal such as a square wave or other periodic signal) to the supply electrode lead 70a of the supply electrode 70. The time-varying signal typically has an offset, such as a DC offset, such that the signal does not drop to zero volts or less than zero volts with respect to ground. Further, the maximum potential of the time-varying signal is less than the potential that can cause an aggregation reaction or a harmful chemical reaction in the sample liquid (e.g., a blood sample). An exemplary time-varying signal is a square wave having a peak-to-peak magnitude between 0.25 and 0.6 volts and a DC offset between 0.5 and 1.5 volts.

[0158] Next, the reader 111 proceeds to a step of monitoring electrical signals present in the distal peripheral portions 102 of the filling electrode lead wires 48a, 52a, 56a, 72a of the filling electrodes 48, 52, 56, 72. When there is no sample liquid in the microchannel network 18, since the supply electrode 70 and the filling electrodes 48, 52, 56, 72 are not in a conductive state, the electrical supply signal is not output by the filling electrode lead wires 48a, 52a, 56a, 72a. On the other hand, when the strip 10 is appropriately filled with the sample liquid as described above, the sample liquid occupies a portion of the microchannel network 18 between the supply electrode 70, the first filling electrode 48 (in the analysis channel 26), and the hematocrit filling electrode 72 (in the hematocrit channel 28). In this state, the sample liquid electrically connects the supply electrode 70 and the filling electrodes 48, 72, and the reader 111 senses the electrical supply signal at the respective contacts of the lead wires 48a, 72a. Based on the sensed electrical supply signal, the reader 111 confirms that the strip 10 is appropriately filled with the sample liquid. When the target determination continues, the reader 111 continues to monitor the electrical supply signal at the filling electrode lead wires 48a, 72, and monitors whether and when the electrical supply signal appears at the filling electrode lead wires 52a, 56a, 72a as expected in response to the movement of the sample liquid induced by the piezoelectric actuator, thereby confirming the appropriate filling and operation of the strip 10 (e.g., the appropriate position and movement at the appropriate timing of the sample liquid in the microchannel network 19).

[0159] One sample liquid is applied to strip 10, and with strip 10 properly filled, sample application zone 20 (port 36) remains in gas communication with the ambient atmosphere 38. The proximal gas-liquid interface of the sample liquid (i.e., the gas-liquid interface closest to sample application zone 20 and in direct gas communication with sample application zone 20) maintains the same gas pressure as the gas pressure of the ambient atmosphere 38 surrounding reader 111 and microfluidic strip 10. Since gas bladder 60 remains sealed with respect to the ambient atmosphere 38, the gas pressure inside gas bladder 60, the portion of microchannel network 18 distal to the distal gas-liquid interface of the sample liquid in analysis channel 26 (i.e., the gas-liquid interface located away from sample application zone 20 by the sample liquid), is higher than the gas pressure of the ambient atmosphere 38 surrounding the strip, but only by an amount just sufficient to overcome the viscous resistance exerted by the interaction between the sample liquid and the inner sidewalls 30 and upper and lower surfaces 12a', 14a' of microfluidic channel network 18. Without compression or decompression of gas bladder 60 by the piezoelectric actuator, the only source of gas pressure distal to the distal liquid-gas interface 98 of the sample liquid exceeding the gas pressure of the ambient atmosphere 38 results from a slight pressure built up (accumulated) even further distal to the distal liquid-gas interface 98 resulting from the capillary action flow of the sample liquid along analysis channel 26. The gas pressure in gas bladder 60 exceeding such a slight (only) excess pressure would propel the sample liquid towards sample application zone 20 (port 36). In the case of a gas pressure in gas bladder 60 below such an excess pressure (such as occurs during decompression of gas bladder 60), the gas pressure exerted by the ambient atmosphere 38 would force the sample liquid to move distally until the pressures equalize again.

[0160] After receiving a signal that the sample liquid has reached the hematocrit filling electrode 72 in the hematocrit channel 28, the reader 111 activates the cyan and IR (infrared) LEDs and the opposing photodiodes to determine the hematocrit of the sample liquid as described above. If the hematocrit exceeds a predetermined limit value, the reader 111 displays an error via the touch screen 115 and interrupts the target determination. The reader 111 also activates the LEDs to determine whether the absolute absorption of the sample liquid matches that of whole blood, or whether absorption (e.g., below a certain limit value) indicates that a non-whole blood sample such as plasma has been applied to the strip. If the hematocrit and absorption are within the predetermined limit values, the reader 111 continues the determination.

[0161] If the determined hematocrit is within the predetermined limit value and the distal liquid-gas interface 98 of the sample liquid has reached the capillary stop 42, the reader 111 activates the flow controller for a time T movReduce the compression of the upper wall portion 78 above the gas bladder 60 therebetween. The actuating end 121 of the piezoelectric bender retreats in the vertical direction. The upper wall portion 78 (under tension below the lower surface 133 of the actuating foot 127) retreats further from the opposing lower substrate 14, increasing the volume of the gas bladder 60 and reducing the gas pressure within the portion of the analysis channel 26 disposed distally of the distal liquid-gas interface 98 of the sample liquid. When the distal gas pressure is reduced, the gas pressure exerted by the ambient atmosphere 38 via the port 36 on the proximal gas-liquid interface of the sample liquid overcomes the resistance forming the capillary stop 42 and the viscous resistance of the sample liquid, forcing the sample liquid to move distally along the analysis channel 26 towards the first reagent zone 44 and the gas bladder 60. The actuation of the piezoelectric bender is calibrated to reduce the pressure within the gas bladder 60 at a rate sufficient to cause the portion of the sample liquid disposed away from the sidewall 30 and the inner surfaces 12a', 14a' to flow at a constant rate of 1.3 mm / s (about 96 nL / s) towards the first reagent zone 44 along the analysis channel 26 and into the first reagent zone 44. However, adjacent to the sidewall 30 and the upper and lower surfaces 12a', 14a' of the analysis channel 26, the sample liquid flows at a lower rate due to the viscous drag force exerted on the sample liquid by these walls and surfaces. Accordingly, the distal liquid-gas interface 98 assumes a parabolic shape, having a maximum velocity at the center of the analysis channel 26 disposed away from any wall or surface and a lower velocity adjacent to the wall 30 and the upper and lower surfaces 12a', 14a'. When the distal liquid-gas interface 98 of the sample liquid passes through each side cavity 46 within the first reagent zone 44, the sample liquid and the gas trapped within the side cavity 46 form a gas-liquid interface 96 at the opening 68 of the side cavity 46 to the analysis channel 26. When the sample liquid enters the first reagent zone 44, the sample liquid solubilizes the lysis reagent 62, which initiates the lysis of the cells within the sample liquid and releases the targets present therein.

[0162] While the actuating end 121 and the actuating leg 127 are retracting in the vertical direction, the leader 111 also causes the piezoelectric actuator to impart a secondary oscillatory motion to the actuating end 121 and the actuating leg 127. Specifically, during time T osc the piezoelectric actuator causes the actuating end 121 to oscillate along the axis a3 with an acoustic frequency between, for example, about 500 Hz and about 2000 Hz and a full-cycle displacement between about 7 μm and about 70 μm while retracting. When the actuating end 121 retracts vertically during the rocking cycle, the pressure applied to the upper surface 131 of the actuating leg 127 by the actuating end 121 is reduced, allowing the actuating leg 127 to move vertically along the axis a3. The upper wall portion 78 retracts vertically relative to the lower surface 133 of the actuating leg 127 to drive the actuating leg 127 vertically along the axis a3. When the actuating end 121 extends downward during the rocking cycle, the pressure applied to the upper surface 131 of the actuating leg 127 by the actuating end 121 increases to drive the actuating leg 127 downward along the axis a3. The lower surface 133 of the actuating leg 127 drives the actuating leg 127 downward along the axis a3. The oscillation of the actuating end 121 causes the upper wall portion 78 of the gas bladder 60 to oscillate, imparting a gas pressure pulse to the gas bladder 60 at essentially the same oscillation frequency.

[0163] As described above, the gas bladder 60 including the contact portion 88 of the outer surface 12b of the upper wall portion 78 contacted by the lower surface 133 of the actuating foot 127 is disposed distally spaced from the portion of the analysis channel 26 occupied by the sample liquid (or any other liquid) during operation of the microfluidic strip 10. During target determination, the portion of the analysis channel 26 (including the gas bladder 60) disposed distally from the distal liquid-gas interface 98 of the sample liquid is occupied by gas and not by the sample liquid or any other liquid. If there is liquid in such a distal portion of the analysis channel 26, it would be an insufficient amount to transmit the pressure oscillations in the gas bladder 60 occupied by gas to the distal liquid-gas interface 98 of the sample liquid. Thus, the effect of the vibration of the piezoelectric bender 117 on the upper wall portion 78 is transmitted indirectly, via the gas bladder 60 occupied by gas and other distal portions of the analysis channel 26, to the distal liquid-gas interface 98 of the sample liquid. Rather than by direct transmission to the sample liquid by vibration or other shock to the portion of the strip 10 occupied by the sample liquid, such as portions of the upper and lower substrates 12, 14.

[0164] The gas pressure pulse impinges on the distal liquid-gas interface 98 of the sample liquid, causing pressure oscillations in the sample liquid. For example, the pressure oscillation in the gas bladder 60, peak-to-peak (((P max -P min ) / P avg )×100) can be between about 5% and about 20%, where P max is the maximum gas pressure during the vibration cycle, P min is the minimum gas pressure during the vibration cycle, and P avg is the average gas pressure during the vibration cycle. The gas pressure oscillation, peak-to-peak (P max -P min) can be, for example, at least about 5 kPa and up to about 200 kPa. The gas pressure oscillations of the gas adjacent to the distal liquid-gas interface of the sample liquid are at too high a frequency, such as an acoustic frequency, for the sample liquid to respond as a substantial bulk movement along the longitudinal axis of the analysis channel during a particular oscillation. For example, independent of the bulk movement of the sample liquid induced by the retraction of the actuating foot 127, the position of the distal liquid-gas interface of the sample liquid can remain essentially the same along the analysis channel 26 during a particular oscillation. Instead, the pressure oscillations in the sample liquid cause pressure oscillations in the gas trapped in the side cavities 46 of the first reagent zone 44 and oscillations of the gas-liquid interface at each side cavity 46. The pressure oscillations in the sample liquid and the pressure oscillations in the gas of the side cavities 46 induce turbulence in the sample liquid. This turbulence has several effects. First, the turbulence promotes solubilization of the dissolved reagent 62 by the sample liquid. Thus, the dissolved reagent 62 is solubilized more efficiently and completely than in the absence of an oscillatory driving flow. Second, the flow increases the rate of bulk transport of the dissolved reagent 62 in the sample liquid, exceeding the rate of diffusion-limited transport in the absence of oscillatory driving transport. This increased bulk transport rate causes the (respective) substances in the sample liquid (e.g., the solubilized dissolved reagent 62 and the target released by lysis of the cells in the sample liquid) to experience different velocities in the flowing sample liquid such that each solubilized substance experiences one similar average velocity. In the absence of an oscillatory driving flow, diffusion-limited transport in the sample liquid is insufficient to transport these substances to regions of different velocities on the time scale of the movement of the liquid into the first reagent zone 44. Thus, in the absence of an oscillatory driving flow, the sample liquid transported into the first reagent zone 44 will exhibit a range of concentrations of such substances in the lateral direction across the width and height of the microchannel. On the other hand, due to the oscillatory driving flow, the (various) substances and the sample liquid are transported more uniformly along the first reagent zone 44, resulting in a more uniform concentration profile of the dissolved reagent 62 and the dissolved target across the width and height of the analysis microchannel 26.

[0165] The vertical retraction and vibration of the actuating end 121 of the piezoelectric bender 117 continue until the distal liquid-gas interface 98 of the sample liquid reaches the first filling electrode 48 at the distal end of the first reagent zone 44. The sample liquid places the supply electrode 70 in conduction with the first filling electrode 48, and at the first filling electrode lead 48a, generates an electrical supply signal indicating that the sample liquid has reached the first filling electrode 48 and completely filled the first reagent zone 44. The piezoelectric actuator actuates the actuating end 121 of the piezoelectric bender 117 to stop the vertical retraction and time T mov ends and maintains the compressed state of the gas bladder 60 at that time. Since the volume of the gas bladder 60 is no longer expanding, the increase in gas pressure distal to the distal liquid-gas interface 98 of the sample liquid stops the sample liquid from flowing further along the analysis channel 26. Time T mov During, the total volume increase of the gas bladder 66 due to the retraction of the actuating foot 127 is substantially the same as the total volume of the analysis channel 26 that is displaced (replaced) by the sample liquid when progressing from the analysis channel vent 40 to the first filling electrode 48. Depending on the volume displaced (replaced), the total vertical retraction of the upper wall portion 78 above the gas bladder 60 is about 15 - 40 μm along the axis a3.

[0166] At a predetermined time after stopping the vertical retraction, the piezoelectric actuator actuates the actuating end 121 of the piezoelectric bender 117 to stop the vibration and time T osc ends and causes the sample liquid to maintain a stopped state within the first reagent zone 44. The sample liquid and the solubilized first reagent 62 are allowed to incubate (cultivate) for a certain time. During this time, the lysis of the target-containing cells in the sample liquid is completed.

[0167] After the incubation (lysis) within the first reagent zone 44 is completed, the reader 111 actuates the flow controller again for a second time T movFurther reduce the compression of the upper wall portion 78 above the gas bladder 60. The actuating end 121 of the piezoelectric bender further retreats in the vertical direction. The upper wall portion 78 (under tension below the lower surface 133 of the actuating foot 127) further retreats from the opposing lower substrate 14, increasing the volume of the gas bladder 60 again and reducing the gas pressure in the portion of the analysis channel 26 located distal to the distal liquid-gas interface 98 of the sample liquid. When the distal gas pressure decreases, the gas pressure exerted by the ambient atmosphere 38 via the port 36 on the proximal gas-liquid interface of the sample liquid overcomes the resistance generated by the gas pressure distal to the distal liquid-gas interface 98 of the sample liquid again, forcing the sample liquid to move distally along the analysis channel 26 towards the second reagent zone 50 and the gas bladder 60. The operation of the piezoelectric bender causes the portion of the sample liquid located at the center of the analysis channel 26 (i.e., the portion of the sample liquid located away from the side walls 30 and the inner surfaces 12a', 14a') to flow along the analysis channel 26 towards the second reagent zone 50 and into the second reagent zone 50 at a sufficient speed to flow at a constant speed of 1.3 mm / s. The pressure in the gas bladder 60 is calibrated to be reduced. When the sample liquid carrying the target enters the second reagent zone 50, the sample liquid solubilizes the labeled binding reagent 64 (along with its fluorescent label), which begins to bind to the target and form the first complex.

[0168] While the actuating end 121 and the actuating foot 127 are retreating in the vertical direction, the reader 111 again causes the piezoelectric actuator to impart a secondary oscillatory motion to the actuating end 121 and the actuating foot 127. Specifically, for a second time period T oscDuring this period, while the piezoelectric actuator is causing the working end 121 to retract, it vibrates along the axis a3 at an acoustic frequency between, for example, about 500 Hz and about 2000 Hz with a full-cycle displacement between about 7 μm and about 70 μm. The vibration induces the same effects as described above with respect to increased dissolution in the side cavities 46 when the sample liquid flows from the first reagent zone 44 towards and into the second reagent zone 50 (i.e., the rate and efficiency of solubilization of the labeled binding reagent 64 are increased), and with respect to the increased rate and uniformity of transport of substances in the sample liquid across the width and height of the analysis channel 26. The increased bulk transport rate in the sample liquid increases the likelihood that the solubilized labeled binding reagent 64 and the target will encounter and bind to each other to form a first complex. Thus, the degree and uniformity of formation of the first complex between the labeled binding reagent 64 and the target are higher than in the absence of the vibration-driven flow.

[0169] The vertical retraction and vibration of the working end 121 of the piezoelectric bender 117 continue until the distal liquid-gas interface 98 of the sample liquid reaches the second filling electrode 52 at the distal end of the second reagent zone 50. The sample liquid places the supply electrode 70 in conduction with the first filling electrode 48 and generates an electrical supply signal at the second filling electrode lead 52a indicating that the sample liquid has reached the second filling electrode 52 and completely filled the second reagent zone 50. The piezoelectric actuator causes the working end 121 of the piezoelectric bender 117 to stop the vertical retraction and end the second time T mov and maintain the compressed state of the gas bladder 60 at that point. Since the volume of the gas bladder 60 is no longer expanding, the increase in gas pressure distal to the distal liquid-gas interface 98 of the sample liquid stops the sample liquid from flowing further along the analysis channel 26. Time T movDuring this period, the total volume increase of the gas bladder 66 due to the retraction of the working foot 127 is substantially the same as the total volume of the analysis channel 26 that is displaced (replaced) by the sample liquid when progressing from the first filling electrode 48 to the second filling electrode 52. According to the volume to be displaced (replaced), the total vertical retraction of the upper wall portion 78 above the gas bladder 60 is about 15 - 40 μm along the axis a3.

[0170] At a predetermined time after stopping the vertical retraction, the piezoelectric actuator operates the operating end 121 of the piezoelectric bender 117 to stop the vibration and end the second time T osc and maintain the state where the sample liquid has stopped within the second reagent zone 50 (excluding the vibration-induced flow within the sample liquid). The sample liquid and the solubilized first reagent 62 are allowed to incubate for a certain time. During this time, the formation of the first complex between the labeled binding reagent 64 and the target, which started as the sample liquid in which the first labeled binding reagent 64 was solubilized, is completed.

[0171] After the incubation (formation of the first complex) within the second reagent zone 50 is completed, the reader 111 operates the flow controller again to further reduce the compression of the upper wall portion 78 above the gas bladder 60 during the third time T mov The operating end 121 of the piezoelectric bender retracts further in the vertical direction. The operation of the piezoelectric bender is calibrated to reduce the pressure within the gas bladder 60 at a speed sufficient to cause the distal liquid-gas interface 98 of the sample liquid (i.e., the portion of the sample liquid disposed away from the side wall 30 and the inner surfaces 12a', 14a') disposed at the center of the analysis channel 26 to flow along the analysis channel 26 towards the detection zone 54 and into the detection zone 54 at a constant speed of 1.3 mm / s. When the sample liquid accompanying the first complex enters the detection zone 54, the sample liquid solubilizes the magnetic binding reagent 66 (accompanying its magnetic particles), which begins to bind to the first complex (including the labeled binding reagent 64 and the target) to form a second complex.

[0172] While the actuating end 121 and the actuating leg 127 are retracting in the vertical direction, the leader 111 again acts on the piezoelectric actuator to impart a secondary oscillatory motion to the actuating end 121 and the actuating leg 127. Specifically, during the third time period T osc while the piezoelectric actuator acts on the actuating end 121 and retracts it, for example, at an acoustic frequency between about 500 Hz and about 2000 Hz and with a full-cycle displacement between about 7 μm and about 70 μm, it vibrates along the axis a3. The vibration induces the same effects as described above with respect to the side cavities 46 when the sample liquid flows from the second reagent zone 50 towards and into the detection zone 54, namely an increased dissolution (i.e., an increased rate and efficiency of solubilization of the magnetic binding reagent 66), and an increased rate and uniformity of transport of substances (such as the first complex) within the sample liquid across the width and height of the analysis channel 26. The increased bulk transport rate within the sample liquid also increases the likelihood that the solubilized magnetic binding reagent 66 and the first complex will bind to form the second complex. Thus, the degree and uniformity of formation of the second complex are higher than in the absence of the vibration-driven flow.

[0173] The vertical retraction and vibration of the actuating end 121 of the piezoelectric bender 117 continue until the distal liquid-gas interface 98 of the sample liquid reaches the third filling electrode 56 at the distal end of the detection zone 54. The sample liquid places the supply electrode 70 in electrical communication with the third filling electrode 56 and generates an electrical supply signal at the third filling electrode lead 56a indicating that the sample liquid has reached the third filling electrode 56 and completely filled the detection zone 54. The piezoelectric actuator stops the vertical retraction of the actuating end 121 of the piezoelectric bender 117 and ends the third time period T mov and maintains the compressed state of the gas bladder 60 at that point. Since the volume of the gas bladder 60 is no longer expanding, the increase in gas pressure distal to the distal liquid-gas interface 98 of the sample liquid stops the sample liquid from flowing further along the analysis channel 26. Time period T movDuring this period, the total volume increase of the gas bladder 66 due to the retraction of the working foot 127 is substantially the same as the total volume of the analysis channel 26 that is moved (replaced) by the sample liquid when progressing from the second filling electrode 52 to the third filling electrode 56. According to the volume to be moved (replaced), the total vertical retraction of the upper wall portion 78 above the gas bladder 60 is about 15 - 40 μm along the axis a3.

[0174] At a predetermined time after stopping the vertical retraction, the piezoelectric actuator stops the vibration by actuating the actuating end 121 of the piezoelectric bender 117, and the third time T osc ends, and the sample liquid is maintained in a state of being stopped within the detection zone 54 (excluding the vibration-induced flow within the sample liquid). The sample liquid accompanied by the first complex and the solubilized magnetic binding reagent 66 is allowed to be incubated for a certain time. During this time, the formation of the second complex between the first complex and the magnetic binding reagent 64, which started as the sample liquid that first solubilized the magnetic binding reagent 66, is completed.

[0175] After the incubation within the detection zone 54 is completed, the reader 111 actuates the magnetic field generator to move the magnetic field generator from the first position to the second position, whereby the second complex containing the magnetic particles of the second reagent 66 is forced to move relative to the inner surface 14a' of the lower substrate 14 by an amount sufficient to retard the movement of the second complex in the presence of the bulk movement of the sample liquid.

[0176] When the magnetic field generator is moved to the second position, the reader 111 again actuates the flow controller to remove the sample liquid, the unbound (non-complex-forming) labeled binding reagent 66, and other accompanying substances that may increase the background signal during the detection process, from the detection zone 54. During the fourth time T mov as described in the process where the piezoelectric flow controller first compresses the gas bladder 60 before applying the sample liquid to the strip 10, the piezoelectric bender 117 is actuated to press the lower surface 129 of the actuating end 121 against the upper surface 131 of the working foot 127, increasing the compression of the gas bladder 60.

[0177] For the sample liquid disposed within the analysis channel 26 between the application zone 20 (port 36) and the gas bladder 60, the increased compression (reduced volume) of the gas bladder 60 increases the gas pressure exerted by the gas within the gas bladder 60 on the distal liquid-gas interface 98 of the sample liquid, thereby overcoming the viscous resistance of the sample liquid and the gas pressure of the ambient atmosphere acting on the proximal gas-liquid interface of the sample liquid, and driving the distal gas-liquid interface (and the proximal portion of the sample liquid) from the detection zone 54 towards the sample operating port 36. The distal gas-liquid interface (and the proximal portion of the sample liquid) is driven proximally at least near the location of the analysis channel vent 40.

[0178] The rate of vertical compression of the gas bladder 60 by the piezoelectric bender 117 is calibrated to increase the gas pressure acting on the distal liquid-gas interface 98 of the sample liquid at a rate sufficient to cause the portion of the sample liquid disposed at the center of the analysis channel 26 (i.e., the portion of the sample liquid disposed away from the sidewalls 30 and the inner surfaces 12a', 14a') to flow proximally along the analysis channel 26 at a constant rate of 20 μm / s (3.3 nL / s) from the detection zone 54. The flow rate when discharging the sample liquid from the detection zone 54 is slower than the flow rate at which the sample liquid is introduced into the detection zone 54, reducing the tendency for the second complex, along with the sample liquid, unbound labeled binding reagent 64, and other accompanying substances that may increase the background signal during subsequent detection steps, to be disadvantageously discharged.

[0179] While the actuating end 121 and the actuating foot 127 are compressing the upper wall portion 78 on top of the gas bladder 60, the reader 111 causes the piezoelectric actuator to impart a secondary oscillatory motion to the actuating end 121 and the actuating foot 127 as described above. Specifically, at the fourth time T oscDuring this period, the piezoelectric actuator vibrates along the axis a3 with an acoustic frequency between about 500 Hz and about 2000 Hz and a full-cycle displacement between about 7 μm and about 70 μm while actuating the working end 121 to compress the upper wall portion 78. The vibration induces the same effects as described above for the side cavities and for the increased rate and uniformity of mass transport. The degree of turbulence induced by the vibration and the velocity of the bulk flow of the sample liquid induced by the increasing pressure are low enough that the second complex (including the magnetic binding reagent 66) remains immobilized on the inner surface 14a' of the lower substrate within the detection zone 54. On the other hand, the turbulence and bulk flow induced by the vibration are sufficient to increase the efficiency and uniformity across the height and width of the microchannel, whereby unbound labeled binding reagents (with their detectable labels) are removed from the detection zone 54.

[0180] The compression and vibration continue until the gas bladder 60 reaches an operatively fully compressed state as determined from the calibration signal stored during the initial compression of the gas bladder 60 as described above. After the gas bladder 60 is recompressed and the vertical actuation of the piezoelectric actuator stops (the end of the fourth time T osc ends) and the vibration stops (the end of the fourth time T osc ends), the sample liquid (including unbound labeled binding reagent 64 and other attendant substances) is removed from the second reagent zone and the distal liquid-gas interface 98 is moved proximally to a position approximately at the capillary stop 42. Only the immobilized second complex and a thin film of the remaining sample liquid remain in the detection zone 54. The amount of the remaining second complex indicates the concentration of the target in the sample liquid applied to the sample application zone (port 36). Next, the reader 111 activates an optical detector to detect fluorescence from the detectable label (label) of the second complex. The reader determines the concentration of the target in the sample liquid based on the detected fluorescence.

[0181] Upon completion of the determination, the reader 111 actuates the piezoelectric actuator to completely retract the actuating end 121 of the piezoelectric bender 117 vertically from the upper surface 129 of the actuating foot 127, completely reducing the compression of the gas bladder 60 so that the strip 10 can be removed from the reader 111. The strip 10 is a single-use strip and is discarded after the determination.

[0182] Referring to FIGS. 6 and 7, the microfluidic strip 210 is configured to be used with a diagnostic reader, such as the diagnostic reader 111, in the determination (decision) of the presence and / or amount of a target (e.g., a biomolecule such as a protein) present in a sample liquid applied to the strip 210. The reader 111 also actuates the strip 210 to determine (decide) the physicochemical properties (e.g., hematocrit) of the sample liquid applied to the strip 210. The reader 111 actuates the strip 210 as described for the strip 10.

[0183] The strip 210 includes an upper substrate 212 and a lower substrate 214 each made of a 100-μm-thick polyester film. The lower surface 212a of the upper substrate 212 and the upper surface 214a of the lower substrate 214 are adhered so as to face each other with a 110-μm thickness of the adhesive layer 216. The adhesive layer 216 is partially absent, for example removed, and defines a microfluidic channel network 218 between the opposing surfaces 212a, 214a of the upper and lower substrates 212, 214. The microfluidic channel network 218 has a sample application zone 220, a common supply channel 222, a branch channel 224, an analysis channel 226, and a hematocrit channel 228. The microfluidic channel network 218 has side walls 230 defined by the adhesive layer 216, an upper wall 232 defined by a portion of the upper substrate 212 located above the absent portion of the adhesive layer 216, and a lower wall 234 defined by a portion of the lower substrate 214 located below the absent portion of the adhesive layer 216. The upper wall 232 has an inner surface 212a' defined by a portion of the surface 212a exposed by the absent portion of the adhesive layer 216. The lower wall 234 has an inner surface 214a' defined by a portion of the surface 214a exposed by the absent portion of the adhesive layer 216. The upper substrate 212 has an outer (upper) surface 212b, and the lower substrate 214 has an outer (lower) surface 214b.

[0184] The sample liquid applied to port 236 of sample application zone 220 flows, by capillary action as described for strip 10, along common supply channel 222 to branch channel 224, and then to analysis channel 226 and hematocrit channel 228. In strip 210, common supply channel 222 is tapered to enhance the capillary force moving the liquid in the distal direction and has a width that decreases as it progresses distally from port 236. Other than the tapered common supply channel 222, the dimensions of the elements of microfluidic network 218 are similar (e.g., may be the same) to the dimensions of the elements of microfluidic network 18 of strip 10. Port 236 places the channels of channel network 218 in gas communication with the gas of the ambient atmosphere 238, such as air. Gas bladder 260 is the distal end of microfluidic channel network 218 and is in gas communication with ambient atmosphere 238 via port 236, hematocrit channel vent 276, and analysis channel vent 240 as described for gas bladder 60 of strip 10. The portion of upper wall 232 located above gas bladder 260 defines gas bladder upper wall 278, and the portion of lower wall 234 located below gas bladder 260 defines gas bladder lower wall 284.

[0185] Hematocrit channel 228 is configured and operates in a manner similar to hematocrit channel 28 to facilitate reagent-free optical determination of the hematocrit of the liquid sample of blood applied to sample application zone 220.

[0186] The analysis channel 226 is configured and arranged to facilitate determination of the presence and / or amount of a target present in the sample liquid. The analysis channel 226 includes, proceeding distally from the branch channel 224 along the longitudinal axis a of the analysis channel 226, an analysis channel vent 240, a capillary stop 242, a first reagent zone 244, a plurality of side cavities 246, a first filling electrode 248, a second reagent zone 250, a second filling electrode 252, a detection zone 254, a third filling electrode 256, a spacer channel 258, and a gas bladder 260.

[0187] As described for the strip 10, the electrodes of the strip 210 are configured and arranged to allow the reader 111 to monitor (monitor) the proper filling of the strip 210 with sample liquid, the proper movement of the sample liquid within the strip 210, and the operation (e.g., compressed state) of the gas bladder 260. Each of the supply electrode 270 and the filling electrodes 248, 252, 256, 272 is disposed on the inner surface 212a' of the upper wall 232 at a position where the sample liquid within the microchannel network 218 contacts the electrode. Each of the electrodes is connected to the distal peripheral edge 302 of the strip 210 to engage a corresponding contact (not shown) within the reader 111 via its respective lead wire. A portion of the lead wire 248a of the first filling electrode 248 and a portion of the lead wire 256a of the third filling electrode 256 pass along the inner surface 212a' of the gas bladder upper wall 278, defining first and second intervening conductive lead electrodes 248a', 256a' respectively. A conductive bridge contact 286 is disposed on the inner surface 214a' of the gas bladder lower wall 284 and is positioned below the lead electrodes 248a', 256a'. As described for the gas bladder 60 of the strip 10, when the gas bladder 260 is fully compressed, the bridge contact 286 and the lead electrodes 248a', 256a' operate to sense.

[0188] Referring further to FIGS. 8 and 9, the first reagent zone 244 contains the lysis reagent 62, the second reagent zone 250 contains the labeled binding reagent 64, and the detection zone 254 contains the magnetic binding reagent 66. The upper surface 214a of the lower substrate 214 includes a first reagent deposition boundary 304, a second reagent deposition boundary 306, and a detection reagent deposition boundary 308 that respectively correspond to the first reagent zone 244, the second reagent zone 250, and the detection zone 254, where the reagents 62, 64, 66 are respectively deposited. The deposition boundaries 304, 306, 308 are defined by a hydrophilic material, such as a hydrophilic coating or hydrophilic layer like the ink printed on the upper surface 214a. Each of the deposition boundaries 304, 306, 308 has a length along the longitudinal axis a21 of the analysis channel 228 that is substantially the same as the corresponding first, second, and detection zones 244, 250, 254, and has a width along an axis a22 that is substantially perpendicular to the longitudinal axis a21 and is greater than the width of the analysis channel 228 within each of the zones 244, 250, 254. In an embodiment of the strip 210, the width of each deposition boundary 304, 306, 308 is 1.5 mm, and the width of the analysis channel 228 is 0.8 mm.

[0189] During manufacturing, each of the reagents 62, 64, 66 is typically deposited in a liquid state within the corresponding deposition boundaries 304, 306, 308. During deposition, the reagent spreads over the upper surface 214a so as to cover most, for example essentially all, of the portion of the upper surface 214a within each deposition boundary 304, 306, 308. The reagent is then dried, for example freeze-dried, if it was deposited in a liquid rather than a non-liquid state. When drying is complete, the adhesive layer 216 is brought into contact with the upper surface 214a of the underlying substrate 214a. As described above, the sidewalls 230 of the microfluidic channel network 218 (including the analysis channel 228) are defined by the adhesive layer 216, and the inner surface 214a' of the microfluidic channel network 218 (including the analysis channel 226) is defined by the portion of the surface 214a that is exposed by a non-present portion, for example a removed portion, of the adhesive layer 216. Since the width of each deposition boundary 304, 306, 308 is greater than the width of the analysis channel 226, at least the intervening portion 62a of the first reagent 62 is interposed outside the analysis channel 226 between the upper surface 214 of the underlying substrate 214 and the adhesive layer 216 thereon. At least a portion of the intervening portion 62a of the first reagent 62 is disposed between adjacent (side) cavities 246 along an axis that is substantially parallel to the longitudinal axis a21 of the analysis channel 226. The width wl of the intervening portion 62a along the axis a22 between the wall 230 and the deposition boundary 304 depends on both the width of the analysis channel 226 and the width of the deposition boundary 304, and such width may be different on one side of the channel as compared to such width on the opposite side of the channel. Independently, on each side of the channel, the width wl can be at least about 50 μm, at least about 100 μm, at least about 150 μm, or at least about 200 μm, and the width wl can be about 500 μm or less, about 400 μm or less, or about 300 μm or less.

[0190] When the reagent 62 is deposited on the upper surface 214a in a state where the adhesive layer 216 is already adhered to the upper surface 214, the reagent can exude (wick) through the opening 268 of the side cavity 246 by capillary action, displace the gas therein, and / or inhibit the opening 268, and thus inhibit the formation of the gas-liquid interface in the presence of the sample liquid (for example, such formation has been described with respect to the side cavity 46 of the strip 10), and reduce or eliminate the benefit of mixing provided by the side cavity 246 during the oscillation of the distal liquid-gas interface of the sample liquid disposed within the analysis channel 226.

[0191] As seen in FIG. 9, the dissolved reagent 62 disposed within the analysis channel 226 on the exposed surface 214a' within the first reagent zone 244 and disposed within the side cavity 246 of the analysis channel 226 forms a thin, uniform distribution layer having a dimension dl along an axis a23 oriented perpendicular to the plane defined (prescribed) by the axis a21, the axis a22, and the lower substrate 214. The thin layer of the reagent 62 readily solvates in the presence of the sample liquid. Further, the intervening reagent 62a disposed outside the analysis channel 226 on the lower surface 214a located beneath the adhesive layer 216 also forms a thin layer having the dimension d1. The gap between the lower surface 216a of the adhesive layer 216 and the upper surface 214a of the lower substrate 214 is narrow enough to prevent the sample liquid from exuding (wicking) between them to an extent that would cause a loss of sample liquid sufficient to endanger either the integrity of the strip 210 or the performance of the assay performed using its analysis channel 226. The reagents 64, 66 are similarly deposited within the deposition boundaries 306, 308 and form an intervening portion beneath the adhesive layer 316 as described for the dissolved reagent 62.

[0192] When the manufacture of strip 210 is completed, strip 210 does not contain liquid as described for strip 10, and the only liquid applied to strip 210 in use is the sample liquid containing the target to be determined. Strip 210 is configured not to require the introduction of liquids other than the sample liquid containing the target to be determined, for example, it is not configured to allow it.

[0193] Here, referring to FIGS. 10 and 11, one embodiment of the analysis channel 326 of the microfluidic strip includes a filling electrode 348 and first and second hydrophobic patches 348b', 348b" that cover all but the central portion 348' of the filling electrode 348a. The central portion 348' of the filling electrode 348 remains exposed to the sample liquid passing along the analysis channel 326 and functions as described for the charging electrodes of strips 10 and 210 to sense the presence of liquid therein. Each hydrophobic patch 348b', 348b" is formed from a hydrophobic layer (e.g., hydrophobic ink) and preferably has a contact angle with deionized water determined using the sessile drop technique with a contact angle goniometer of at least about 75°, at least about 80°, or at least about 85°. The filling electrode 348 is connected by a lead wire 348a to the distal periphery of a microfluidic strip (not shown). The filling electrode 348 can be used in association with a source electrode as described for microfluidic strips 10, 210. FIGS. 10 and 11 illustrate only a single filling electrode, but the analysis channel 326 can include a plurality of filling electrodes each having the same characteristics as the filling electrode 348. Those charging electrodes can be spaced along the longitudinal axis of the analysis channel and can be spaced, for example, by one or more reagent zones as described for the analysis channels 26, 226 of strips 10, 210.

[0194] The analysis channel 326 is defined by the wall 330 of the adhesive layer 316, the surface 313a' of the lower substrate 314, and the surface of the upper substrate, which is not shown for clarity. The wall 330 includes opposing first and second notches 330', 330", which are substantially aligned with the electrodes 348. The notches 330', 330" increase the surface area of the first and second hydrophobic patches 348b', 348b" available for contact with the sample liquid within the analysis channel 326, even in cases where manufacturing tolerances cause slight misalignment of various features. The analysis channel 326 also includes a plurality of side cavities 346, each having an opening 368, as described for the side cavities 46, 246 of the strips 10, 210.

[0195] The analysis channel 326 has a width w2 of approximately 800 μm along a transverse axis a32 perpendicular to the longitudinal axis a31 of the analysis channel 326. Each hydrophobic patch 348b', 348b" extends a distance d2 of approximately 280 μm from the adjacent wall 330 along the transverse axis a32 and extends a length l1 of 500 μm along the longitudinal axis a31 on each side of the filling electrode 348. The hydrophobic patches 348b', 348b" are spaced apart from each other by a distance d4 of approximately 250 microns along the transverse axis a32. Each notch 330', 330" has a length l2 of approximately 1070 μm along the longitudinal axis a31 and a depth d5 of approximately 530 μm along the transverse axis a32. The electrode 348 has a width w3 of approximately 400 μm along the longitudinal axis a31.

[0196] In practice, one or more fill electrodes 348, e.g., one or more fill electrodes 348 with hydrophobic patches 348b', 348b" and / or notches 330', 330", can be used with microfluidic strips such as strips 10, 210, and readers such as reader 111. When a sufficient amount of sample liquid is applied to the strip and the strip functions properly, the distal liquid-gas interface of the sample liquid moving distally along analysis channel 326 contacts the central portion 348' of fill electrode 348 to establish electrical continuity with the source electrode of the strip. A time-varying signal applied (imposed) to the source electrode is detected by the reader at lead 348a, indicating the presence of sample liquid at the location of fill electrode 348 within analysis channel 326. After determining that the sample liquid is in contact with central portion 348', the reader can stop the movement of the sample liquid. Thereafter, the reader can reverse the movement of the sample liquid to move it proximally along analysis channel 326. When the liquid-gas interface of the sample liquid moves proximally to the fill central portion 348' of fill electrode 348, the hydrophobic patches 348b', 348b" ensure dewetting of the central portion 348' so that the remaining film of liquid does not maintain electrical continuity between the source electrode and central portion 348'. Thereby, the reader determines that the time-varying signal from the source electrode is no longer detected at fill electrode 348, indicating that the sample liquid has been withdrawn therefrom.

[0197] As described above, the analysis channel 326 may include a plurality of filling electrodes having the characteristics of the filling electrode 348. The reader may continue to move the sample liquid until the distal liquid-gas interface of the sample liquid moves proximally to the second filling electrode within the analysis channel 326. When the second filling electrode becomes de-wetted, the conduction between the second filling electrode and the source electrode is interrupted, and the signal indicating the conduction is stopped. Then, the reader may stop the movement of the sample liquid and may be in a state where the sample liquid is moved by an accurate known proximal direction distance determined by the separation (interruption) of the filling electrodes along the longitudinal axis a31 within the analysis channel 326. Thereafter, the reader may reverse the direction of movement of the sample liquid again, may move the sample liquid distally again, and may detect signals from the second filling electrode and the charging electrode when the liquid-gas interface moves along the analysis channel 326.

[0198] By detecting signals from such one or more spaced-apart fill electrodes within analysis channel 326, the reader can accurately control and monitor the sample liquid such that the sample liquid repeatedly moves in a first direction (e.g., distal direction) and then in a second direction (e.g., proximal direction). Such movement can move the sample liquid into and through a reagent zone spaced by a pair of fill electrodes to facilitate mobilization (movement) of reagents and / or mixing and / or binding of reagents and targets, and then back from the reagent zone. Such movement allows a larger volume of sample liquid to be moved through the reagent zone or detection zone, thereby allowing exposure of the reagent to a greater number of targets than when only a smaller volume of sample liquid is moved through the detection zone. In zones containing magnetically coupled reagents, a magnet can be used to hold the reagent within the zone such that the reagent binds and aggregates with targets present in the sample liquid at the location of the reagent. In some embodiments, a bound reagent, e.g., immobilized, fixed within a zone can be used and no magnet is utilized to hold the reagent while moving liquid into, through, and back from the zone. Movement of the sample liquid can be effected by increasing or reducing the pressure of the gas adjacent to the distal liquid-gas interface of the sample liquid. The reader can also impart vibrations to the gas pressure, as described for strips 10, 210.

[0199] Next, referring to FIG. 12, the microfluidic strip 510 includes a microfluidic channel network 518 having a sample application zone 520, a common supply channel 522, a common branching channel 524, a hematocrit channel 528, and four analysis channels 526a, 526b, 526c, 526d. The microfluidic strip 510 is used in association with a reader, as described, for example, for the microfluidic strip 10, the microfluidic strip 210, or the analysis channel 326. The microfluidic strip 510 is formed from an upper substrate 512 and a lower substrate 514 adhesively fixed and opposed to each other by an adhesive layer, as described, for example, for the microfluidic strips of the microfluidic strips 10, 210, and the analysis channel 326. The sample application zone 520 is a port 536 that penetrates the upper substrate 512, as described for the ports 36, 236.

[0200] The hematocrit channel 528 is configured and arranged to facilitate the reagent-free optical determination of the hematocrit of a liquid sample of blood, as described for the hematocrit channel 28. In the direction proceeding distally from the branch channel 524, the hematocrit channel 528 includes a supply electrode 570, a hematocrit filling electrode 572, a hematocrit detection zone 574, and a vent channel 576 extending between the hematocrit detection zone 574 and the vent 576a. The vent channel 576 has a length of 15 mm, a height of 110 μm, and a width of 150 μm between the hematocrit detection zone 574 and the vent 576a. The cross-sectional area of the vent channel 576 is small enough to substantially prevent the sample liquid from entering the vent channel. The vent 576a is disposed within the proximal portion of the microfluidic strip 510. In use, the proximal portion of the microfluidic strip including the vent 576a protrudes from the reader. Even if the sample liquid is inadvertently discharged from the vent 576, the sample liquid remains outside the reader and does not contaminate its interior. The sample application zone 520 and the vent 576a may be the only path through which gas can enter and exit the microfluidic channel network 518.

[0201] Each of the analysis channels 526a, 526b, 526c, 526d is configured and arranged to facilitate the determination of the presence and / or amount of at least one target present in the sample liquid applied to the sample application zone 520. Each target determined using each analysis channel may be the same as or different from the targets determined using the other analysis channels. Proceeding distally from the common branch channel 524, each analysis channel 526a, 526b, 526c, 526d begins at its respective proximal origin 526' and includes a first reagent zone 544, a first filling electrode 548, a second reagent zone 550, a second filling electrode 552, a detection zone 554, a third filling electrode 556, a spacer channel 558, and a gas bladder 560. Each analysis channel has a length of approximately 20 mm between the proximal origin 526' and the distal end of the gas bladder 560.

[0202] Within each analysis channel, the filling electrodes 548, 552, 556 each include a respective hydrophobic patch as described for the filling electrode 348 of the analysis channel 326. Within each gas bladder 560, the respective lead wires of the filling electrodes 548, 556 each define a respective intervening lead electrode, and the gas bladder 560 defines corresponding bridge contacts as described for the gas bladder 60. The reagent zones and detection zones of each of the analysis channels 526a, 526b, 526c, 526d can be configured as described for the microfluidic strip 10, the microfluidic strip 210, or the analysis channel 326. Although not shown, each analysis channel can include side cavities as described for the microfluidic strip 10, the microfluidic strip 210, or the analysis channel 326.

[0203] The respective proximal origins 526’ of each analysis channel connect to the branch channel 524 at different positions along it. For each of the plurality of analysis channels, the proximal origin provides the only path through which liquid and gas (vapor) can enter and exit such an analysis channel. The gas bladder 560 of each analysis channel defines its distal end. In use, the distal portion of the microfluidic strip 510 is received within the reader. The distal portion includes at least the gas bladder of each analysis channel and most or all of the remainder of each analysis channel. The reader includes respective flow controllers for each analysis channel as described for the microfluidic strip 10 and the microfluidic strip 210. For example, the flow controller can compress and decompress the gas bladder and release gas from it or draw gas into it. The sample liquid present within the analysis channel is moved distally along the analysis channel towards the gas bladder or proximally away from the gas bladder.

[0204] In use, the microfluidic strip 510 is inserted into the reader, and each flow controller of each channel operates the gas bladder of such an analysis channel to an operatively fully compressed state, as described, for example, for microfluidic strips 10 and 210. As described for microfluidic strips 10 and 210, the reader calibrates the degree of compression required to fully compress the upper wall portion 78 to position each gas bladder 560 in an operatively fully compressed state, and the amount of force required to be applied by the piezoelectric actuator to displace the upper wall portion 78 of each gas bladder 560. In use, the degree of displacement and amount of force required to achieve a given fluid operation may depend on whether the upper walls of one or more other gas bladders of the strip 510 are simultaneously operated (e.g., compressed, decompressed, and / or vibrated). For example, compression of a gas bladder places its upper wall under tension, and other gas bladders of the strip may consequently experience an increase in tension. Thus, the reader may acquire calibration signals for each gas bladder in a first state where no other gas bladders of the strip are simultaneously operated and / or in a second state where one or more other (other) gas bladders of the strip are also operated (e.g., compressed, decompressed, and / or vibrated). For each gas bladder, the reader stores calibration signals for the degree of displacement and amount of force required to achieve a given fluid operation in either or both of the first and second states. During operation of the strip 510, the reader can thus operate the piezoelectric actuator of each gas bladder and operate the gas bladder, regardless of whether one or more other gas bladders of the strip are simultaneously operated.

[0205] Next, the sample liquid is applied to the sample application zone 520. The sample liquid flows by capillary action along the common supply channel 522 until it reaches the branch channel 524. In the branch channel 524, the sample liquid branches into a first portion that travels along the branch channel 524 towards the hematocrit channel 528 and a second portion that travels along the branch channel 524 towards the respective proximal origin 526' of each of the analysis channels 526a, 526b, 526c, 526d. The first portion of the sample liquid travels into the hematocrit channel 528 until the corresponding distal liquid-gas interface of the sample liquid (e.g., the liquid-gas interface of the sample liquid in the hematocrit channel 528 located away from the sample application zone 520 by the divided amount of the sample liquid in the hematocrit channel 528, the common branch channel 524, and the common supply channel 522) fills the hematocrit detection zone 574. As the sample liquid travels along the hematocrit channel 528, gas is moved out of the hematocrit channel and out of the microfluidic network 518 via the vent channel 576 and the vent 576a, but the cross-sectional area (narrowness) of the vent channel 576 substantially prevents the intrusion of the sample liquid. The release of gas through the vent 576a allows the sample liquid to fill the hematocrit channel 528 by capillary action.

[0206] The second portion of the sample liquid proceeds by capillary action along the common branch channel 524. The sample liquid enters each of the analysis channels 526a, 526b, 526c, 526d. Since each analysis channel is sealed with respect to the entry and exit of gas, the gas pressure in front of the sample liquid (i.e., the gas pressure distal to the liquid-gas interface distal to the sample liquid) increases and stops the distal progression of the sample liquid before entering the first detection zone of each analysis channel (i.e., proximally thereto). Subsequently, the reader activates the respective flow controllers of each analysis channel to mix and / or move the sample liquid distally or proximally along the analysis channel, as described, for example, for the microfluidic strip 10, the microfluidic strip 210, or the analysis channel 326. The reader also activates an optical detection system, a magnetic field generator, and the respective flow controllers to detect one or more targets within each analysis channel.

[0207] Here, referring to FIGS. 13A through 13D, the microfluidic strip 610 includes a microfluidic channel network having a sample application zone 620, a common supply channel 622, a common branching channel 624, and four analysis channels 626a, 626b, 626c, 626d extending therefrom. The microfluidic strip 610 is formed from, for example, an upper substrate 612 and a lower substrate 614 adhesively fixed and adhered to face each other by an adhesive layer 616, as described for the microfluidic strips of microfluidic strips 10, 210, 510 and the analysis channel 326. The sample application zone 620 is a port 636 that penetrates the upper substrate 612, as described for ports 36, 236, 536. The microfluidic strip 610 is used with a reader, such as reader 111, and the sample liquid is manipulated (e.g., mixed and / or moved within the microfluidic channel network) as described for, e.g., microfluidic strips 10, 210, 510 or the analysis channel 326, and a target is detected. The reader can activate an optical detection system, a magnetic field generator, and respective flow controllers of the reader to detect one or more targets within each analysis channel.

[0208] The microfluidic channel network of the strip 610 has sidewalls 630 defined by the adhesive layer 616, an upper wall 632 defined by a portion of the upper substrate 612 located above the non-present portion of the adhesive layer 616, and a lower wall 634 defined by a portion of the lower substrate 614 located below the non-present portion of the adhesive layer 616. The upper wall 632 has an inner surface 612a' defined by a portion of the surface 612a exposed by the non-present portion of the adhesive layer 616. The lower wall 634 has an inner surface 614a' defined by a portion of the surface 614a exposed by the non-present portion of the adhesive layer 616. The upper substrate 612 has an outer (upper) surface 612b, and the lower substrate 614 has an outer (lower) surface 614b.

[0209] Proceeding distally from the bifurcation channel 624, each analysis channel has a first hydrophobic stop 611, a first pair of hydrophobic patches 613, a common first filling electrode 672, a first reagent zone 644 having a first pair of reagent deposition boundaries 615, a second filling electrode 648, a second pair of hydrophobic patches 617, a second reagent zone 650 having a second pair of reagent deposition boundaries 619, a third filling electrode 656, a third pair of hydrophobic patches 621, a second hydrophobic stop 623, and a gas bladder 660. Each of the second and third pairs of hydrophobic patches 617, 621 is associated with its respective filling electrode 648, 56 and notch 630' in the sidewall 630 as described for the analysis channel 326. During operation of the strip 610, the second reagent zone 650 is used as the detection zone.

[0210] The reagents in the first and second reagent zones 644, 650 are created and configured to facilitate determination of one or more target and / or control reactions. For example, the reagents can be configured as the reagents of the strips 10, 210, 510, as the reagents of the analysis channel 326, or as the reagents of the strips of Example 1 or 2. The reagents of each analysis channel can be configured to determine the same or different targets as the reagents of one or more other analysis channels of the strip 610. The reagents in each reagent zone 644 are deposited on the lower surface 612a' of the upper substrate 612 between the reagent boundaries 615, and the reagents in each reagent zone 650 are deposited on the lower surface 612a' of the upper substrate 612 between the reagent boundaries 619. The opposing members of each pair of reagent boundaries are spaced 600 μm apart along an axis substantially perpendicular to the longitudinal axis of the analysis channel. The analysis channel is 1.2 mm wide at the location of the reagent boundaries.

[0211] The strip 610 includes optical features that increase the signal relative to the noise of fluorescence detection. For example, each pair of opposing members of the reagent boundaries 615, 619 are separated by a distance less than the distance between the opposing walls 630 of the analysis channel, so that the reagent boundaries act as optical slits and hide the walls 630 from the field of view of the reader's optical detector. The reader directs excitation light into the detection zone through the upper substrate 612 and detects fluorescence from the detection zone through the upper substrate 612. Thus, fluorescence that may be excited or emitted from the adhesive of the walls 630 does not reach the detector, increasing the signal-to-noise ratio of the detection process. As another example of such a feature, the upper surface 614a of the lower substrate 614 includes an opaque diffusive reflective layer 627. A portion 627' of the reflective layer 627 forms the lower inner surface 6a' of the second reagent zone (detection layer) 650 of each analysis channel, where it increases the relative amount of fluorescence detected from the fluorescence emitted by the reagent. The reflective layer can be composed of a composition containing, for example, a metal oxide such as aluminum oxide or zinc oxide, or other materials having a high reflectivity (low absorbance) of light within the bandwidth of the fluorescence to be detected. The upper surface 614a of the lower substrate 614 also includes highly opaque absorptive (light-absorbing) patches 629 disposed between adjacent analysis channels. The absorptive patches 629 have a high absorbance within the bandwidth of the excitation light source and, optionally, also within the bandwidth of the fluorescence to be detected. Thus, the absorptive patches 629 reduce the amount of background fluorescence reaching the detector.

[0212] The strip 610 is configured to allow a reader to monitor and control the operation (e.g., compression state) of each gas bladder 660 of each analysis channel, as described for strip 10, 210, 510, for analysis channel 326, or for the strips of Example 1 or 2 (described later). Within each analysis channel, portions of the lead wires of each of the two filling electrodes pass along the inner surface within the gas bladder of that analysis channel, as described for strips 10 and 210, for example. For example, within analysis channel 626a, the portion of lead wire 648a of the second filling electrode 648 and the portion of lead wire 656a of the third filling electrode 656 pass along the inner surface 612a' of the upper wall 678 of the gas bladder, defining first and second intervening conductive lead electrodes 648a', 656a', respectively. A conductive bridge contact 686 is disposed on the inner surface 614a' of the lower wall 684 of the gas bladder, positioned below the lead electrodes 648a', 656a'. When the gas bladder 660 is fully compressed, as described for the gas bladder 60 of strip 10, for example, the bridge contact 686 and the lead electrodes 648a', 656a' operate to sense.

[0213] For example, as described for strip 10, 210, 510, for analysis channel 326, or for the strips of Example 1 or 2 (described later), strip 610 includes electrodes configured and arranged to allow a reader to monitor (monitor) proper filling of the strip 610 with sample liquid and proper movement of the sample liquid within the strip 610. Strip 610 includes a supply electrode 670, a common first filling electrode 672, and second and third filling electrodes 648, 656 for each analysis channel of strip 610, disposed on the lower surface 612a of the upper substrate 612 and intersecting each channel at the position of the upper wall 632, such that the sample liquid within the microchannel network contacts the electrodes. Each of the electrodes is connected to the distal peripheral portion 602 of the strip 610 for engagement with a corresponding contact (not shown) within the reader via its respective lead wire.

[0214] The supply electrode 670 includes a supply electrode contact 670 disposed at the distal peripheral portion 602 of the strip 610, a supply portion 670 disposed in the branch channel 624, and a supply lead 670 extending to the supply portion 670. The liquid present in the branch channel 624 at the position of the supply portion 670 is configured to be in electrical contact with the supply portion 670. When the strip 610 is received by the reader, a contact (not shown) in the reader is configured to input an electrical signal, such as an electrical "supply" signal (e.g., a time-varying signal such as a square wave or other periodic signal), to the electrode contact 670. Except for the supply portion 670, the supply electrode 670 is disposed outside the microfluidic channel network of the strip 610, and the portion of the supply electrode 670 other than the supply portion 670 is prevented from being in electrical contact with the sample liquid present in the microfluidic network. 2 from the supply electrode contact 670 disposed at the distal peripheral portion 602 of the strip 610 to the supply portion 670 disposed in the branch channel 624 3 and the supply lead 670 extending to the supply portion 670 1 including the supply portion 670 3 such that the liquid present in the branch channel 624 at the position of the supply portion 670 is in electrical contact with the supply portion 670 3 When the strip 610 is received by the reader, a contact (not shown) in the reader is configured to input an electrical signal, such as an electrical "supply" signal (e.g., a time-varying signal such as a square wave or other periodic signal), to the electrode contact 670 2 including the supply portion 670 3 Except for the supply portion 670, the supply electrode 670 is disposed outside the microfluidic channel network of the strip 610, and the portion of the supply electrode 670 other than the supply portion 670 is prevented from being in electrical contact with the sample liquid present in the microfluidic network. 3

[0215] The common first filling electrode 672 includes a filling electrode contact 672 disposed at the distal peripheral portion 602 of the strip 610, a supply lead 672 extending to the first common lead branch portion 672 and the second common lead branch portion 672. The first common lead branch portion 672 extends across the strip 610 in a direction perpendicular to the longitudinal axis of the analysis channels 626a - 626d. A portion 672 of the first common lead branch portion 672 is disposed adjacent to the analysis channel 626a, a portion 672 of the first common lead branch portion 672 is disposed between the analysis channels 626a and 626b, and a portion 672 of the first common lead branch portion 672 is disposed between the analysis channels 626b and 626c. 2 from the filling electrode contact 672 disposed at the distal peripheral portion 602 of the strip 610 to the first common lead branch portion 672 3 and the second common lead branch portion 672 4 and the supply lead 672 extending to the first common lead branch portion 672 and the second common lead branch portion 672 1 including the first common lead branch portion 672 3 The first common lead branch portion 672 extends across the strip 610 in a direction perpendicular to the longitudinal axis of the analysis channels 626a - 626d. 3 A portion 672 of the first common lead branch portion 672 31 is disposed adjacent to the analysis channel 626a 3 A portion 672 of the first common lead branch portion 672 32 is disposed between the analysis channels 626a and 626b 3 A portion 672 of the first common lead branch portion 672 33 ​is disposed between analysis channels 626b and 626c and has a first common lead wire branching portion 672 3 a portion 672 34 is disposed between analysis channels 626c and 626d. The first common lead wire branching portion 672 3 includes liquid sensing portions 672a, 672b, 672c, and 672d respectively disposed within analysis channels 626a, 626b, 626c, and 626d, and sample liquid present within one of the analysis channels at the position of the liquid sensing portion is adapted to be in electrical contact with the liquid sensing portion. The first common lead wire branching portion 672 3 a portion 672 31 and the liquid sensing portion 672a, the first common lead wire branching portion 672 3 a portion 672 32 and the liquid sensing portion 672b, the first common lead wire branching portion 672 3 a portion 672 33 and the liquid sensing portion 672c, and the first common lead wire branching portion 672 3 a portion 672 34 and the liquid sensing portion 672d form continuous sensing pairs. The sensing portions of each sensing pair are disposed within different analysis channels of the microfluidic network of the strip 610.

[0216] The second common lead wire branching portion 672 4 extends to a liquid sensing portion 672e disposed within the common branching channel 624 such that liquid present within the branching channel 624 at the position of the liquid sensing portion 672e is adapted to be in electrical contact with the liquid sensing portion 672e. Except for the liquid sensing portions 672a to 672e, the filling electrodes 672 are disposed outside the microfluidic channel network of the strip 610 such that portions of the filling electrodes 672 other than the liquid sensing portions 672a to 672e are not in electrical contact with the sample liquid present within the microfluidic network.

[0217] Within each reagent zone of each analysis channel of the strip 610, the side wall 630 includes two offset side cavities 646. The side cavities 646 are formed and configured, for example, as cavities 46, 246, 346, to facilitate mixing within each analysis channel. Each side cavity 646 has a width of 120 μm, a length of 900 μm, and a height of 110 μm. Each analysis channel has a width of 1.2 mm and a height of 110 μm. Instead of facing each other as shown, for example, for side cavity 46 in FIG. 3, the side cavities 646 are offset from each other such that each side cavity faces a non-destructive portion of the side wall 630 without a side cavity.

[0218] In use, sample liquid is applied to the sample application zone 620 and flows by capillary action along the supply channel 622 until it reaches the branch channel 624. Along the branch channel 624, a first portion of the sample liquid flows by capillary action to each of the four analysis channels 626a - 626d, and a second portion of the sample liquid flows by capillary action along the branch channel 624 across the liquid sensing portion 672e of the common electrode 672 and further across the supply portion 670 of the supply electrode 670 3 and then stops moving at the proximal end of the narrow vent channel 676. The vent channel 676 terminates at the vent 676a. The vent channel 676 and the vent 676a are sized and configured to operate as described for the vent channel 576 and the vent 576a. Each (each) portion of the sample liquid entering each analysis channel stops moving at the respective capillary stop 611 within each analysis channel. Within each analysis channel, each capillary stop 611 is positioned such that the sample liquid contacts the respective liquid sensing portions 672a, 67b, 672c, 672d of the common electrode 672 disposed within each analysis channel when stopped by the capillary stop 611.

[0219] The reader supplies a time-varying signal, such as the time-varying signal described elsewhere in this specification, for example, in Example 1 and for the reader 111 of the strip 10 and the supply electrode 70, to the supply contact 670 of the supply electrode 670 2 Therein. The reader also determines the presence and amount (e.g., amplitude) of the electrical signal at the filling electrode contact 672 2 When the strip 610 is properly filled with the sample liquid, the sample liquid establishes conduction (continuity) between the supply portion 670 of the supply electrode 670 3 And the common electrode 672 along each of the following five paths: (1) from the supply portion 670 3 To the liquid sensing portion 672e in the branch channel 624 along the branch channel 624, and (2)-(5) from the supply portion 670 3 To the liquid sensing portions 672a, 672b, 672c, 672d of the common electrodes 672 disposed in each of the analysis channels 626a-626d along the branch channel 624 and along the proximal portions of each of the analysis channels 626a-626d. The reader determines whether the proximal portions of the branch channel 624 and the four analysis channels are properly filled with the sample liquid based on the electrical signal determined at the filling electrode contact 672 of the common electrode 672 at the distal peripheral portion 602 of the strip 610 2 Therein. For example, if the sample liquid does not establish conduction (continuity) between the supply portion 670 3 And one or more of the liquid sensing portions 672a, 672b, 672c, 672d, the total impedance between the supply electrode 670 and the common filling electrode 672 will be higher than when the sample liquid establishes conduction (continuity) between the supply portion 670 3 And each of the liquid sensing portions

[0220] During subsequent operations of the sample liquid within each analysis channel, the reader determines the presence of liquid at each of the second and third electrodes 648, 656 of the analysis channel, for example, as described for the electrodes 348 of strip 10, 210, 510 or analysis channel 326. Hydrophobic patches 617, 621 are positioned on each of the electrodes 648, 656 so as to expose the central portion, as described for the hydrophobic patches 348b’, 348b”, providing more efficient dewetting and enabling the presence / absence of the sample liquid to be determined more efficiently during operation of the sample liquid, as described for analysis channel 326. The sidewalls 630 of each analysis channel include notches 630’. The notches 630’ increase the surface area of the hydrophobic patches exposed to the sample liquid, as described for the notches 330’ of analysis channel 326. Based on a failure to properly fill one or more of the analysis channels of strip 610, the reader may invalidate (e.g., terminate) one assay performed within an improperly filled analysis channel and / or invalidate (e.g., terminate) all assays performed using an improperly filled strip.

[0221] Next, referring to FIG. 14, the microfluidic strip 710 includes a microfluidic channel network having a sample application zone 720 with a sample application port 736, a primary common supply channel 722, a primary common branch channel 724, a hematocrit channel 728, and four analysis channels 726a, 726b, 726c, 726d. The microfluidic strip 710 is actuated by a reader, for example, as described for other microfluidic strips or analysis channels disclosed herein. The microfluidic strip 710 is formed from an upper substrate and a lower substrate adhesively fixed so as to face each other by an adhesive layer, for example, as described for other microfluidic strips or analysis channels disclosed herein.

[0222] The primary common branch channel 724 extends to two secondary common supply channels 722', 722" and the hematocrit channel 728. The hematocrit channel 728 includes a supply electrode 770, a common electrode 772, a hematocrit detection zone 774, and a vent 776. The reader activates the hematocrit detection zone 774 to determine the hematocrit of the blood sample as disclosed for other hematocrit detection zones herein.

[0223] Each of the secondary common supply channels 722', 722" extends to respective secondary common branch channels 724', 724", each of which is fluidly connected to respective pairs of analysis channels 726a, 726b and 726c, 726d. Each of the analysis channels 726a - d is configured and arranged to prepare respective plasma samples from the whole blood sample applied to the sample application zone 720 and to determine the presence and / or amount of C - reactive protein in the plasma sample. The arrangement of the primary common branch channel and the secondary common branch channels ensures that the same distance and microchannel volume are traversed by the liquid sample applied to the sample application port 736 and flowing into each of the analysis channels 726a, 726b, 726c, 726d.

[0224] Proceeding distally from the secondary common branch channels 724', 724", each of the analysis channels 726a, 726b, 726c, 726d begins at respective proximal origins 726' and includes a first carbon strip 751a, a first reagent zone 744, a first filling electrode 748, a second reagent zone 750, a second filling electrode 752, a second carbon strip 751b, a detection zone 754, a third filling electrode 756, a spacer channel 758, and a gas bladder 760. Each gas bladder 760 is configured and arranged as described for gas bladder 60. Each of the analysis channels 726a - d is associated with respective vents 740a, 740b, 740c, 740d disposed within the secondary common branch channels 724', 724''. Each vent communicates with the ambient atmosphere (e.g., air) surrounding the strip 710.

[0225] Each first reagent zone 744 contains 0.45 μL of a solution containing 1 mg / ml of phytohemagglutinin E in a trehalose-containing buffer and 0.45 μL of a solution containing 1 mg / ml of soybean agglutinin in a trehalose-containing buffer, which are deposited and dried on the lower side of the upper substrate. Each first reagent zone 744 has a length of 4.95 mm along its longitudinal axis, a width of 1.2 mm perpendicular to the longitudinal axis, a height of 0.11 mm, and a volume of 0.65 μL. Each second reagent zone 750 contains 100 nm streptavidin-coated magnetic particles bound to biotinylated first anti-CRP Fab and fluorescent particles bound to second anti-CRP Fab, which are applied and dried on the upper side of the lower substrate. The first and second Fabs bind to CRP in a sandwich configuration. Each second reagent zone 750 has a length of 3.9 mm along its longitudinal axis, a width of 0.8 mm perpendicular to the longitudinal axis, a height of 0.11 mm, and a volume of 0.34 μL. The reagents in each second reagent zone 750 are deposited within their respective reagent deposition boundaries 704 as described for the reagent deposition boundary 304. Each detection zone 754 contains a mixture of protein blocking components applied and dried on the lower side of the upper substrate. Each detection zone 754 has a length of 2 mm along its longitudinal axis, a width of 0.8 mm perpendicular to the longitudinal axis, a height of 0.11 mm, and a volume of 0.17 μL.

[0226] Each carbon strip 751a, 751b has a length of 500 μm, a height of about 5 μm, and an arithmetic roughness of Sa - 0.8 along the longitudinal axis of each analysis channel 726. Each reagent deposition boundary 704 is formed of printed hydrophobic carbon having the same length (width) and height as the carbon strip.

[0227] The strip 710 can be operated as follows. The strip is inserted into the reader, and the gas bladder for each analysis channel is moved to an operatively fully compressed state as described, for example, for the gas bladder 60 of strip 10. As described for the reader 111, the reader activates a magnetic field generator. Then, a whole blood sample is applied to the application port 736 of the application zone 720. The whole blood sample flows by capillary action along the common supply channel 722 and the primary common branch channel 724, and further from the branch channel 724, a first portion of the whole blood sample flows by capillary action into the hematocrit detection zone 774, and each second portion of the whole blood sample flows by capillary action into the second common branch channels 724', 724" until the respective distal liquid-gas interface of each second portion of the whole blood sample reaches the respective proximal origin 726' of the analysis channel. Each vent 740a-d and the carbon strip 751 act as capillary stops to stop the capillary flow of the whole blood sample with the respective distal liquid-gas interface at the proximal origin of the analysis channel. The presence of the whole blood sample in each secondary common supply channel is determined using the supply electrode 770 and the common electrode 772 as described, for example, for the common electrode 672.

[0228] Next, the reader activates the flow controller to reduce the gas pressure at the distal liquid-gas interface of each whole blood sample, thereby drawing each whole blood sample along its respective analysis channel until the whole blood sample fills each first reagent zone 744 and the distal liquid-gas interface of each whole blood sample reaches the second filling electrode 752. When the second filling electrode 752 is reached, the actuator stops the flow of the whole blood sample. The whole blood within each first reagent zone aggregates and binds to the aggregation reagent therein. After a short incubation, for example, between about 5 and 20 seconds, the actuator begins to oscillate the gas pressure at each distal liquid-gas interface within each analysis channel, causing the whole blood sample to oscillate proximally and distally within each channel. The distal liquid-gas interface of each whole blood sample is oscillated about a distance near the length of the first reagent zone, for example, ± about 5 mm, and through a volume near the volume of the first reagent zone, for example, ± about 0.65 μL. The cycle time for each complete oscillation is between about 1 and 5 seconds, for example, about 2 seconds per oscillation. The movement speed of the distal liquid-gas interface along each analysis channel is between about 1 and 10 mm / second, for example, about 5 mm / second. The oscillation further mixes the whole blood sample within each first reagent zone with the aggregation reagent therein. The oscillation frequency is between about 3 and 20, for example, about 10.

[0229] Upon completion of the vibration, the actuator stops the flow of the whole blood sample mixed (combined) with the agglutination reagent with each distal liquid-gas interface of the whole blood sample within each analysis channel being around the position of the first carbon strip 751a. Next, the actuator begins to reduce the gas pressure of the distal liquid-gas interface, causing each whole blood sample with the mixed agglutination reagent to move distally towards its respective gas bladder 760 within each analysis channel. The moving speed of the distal liquid-gas interface along each analysis channel is about 0.05 - 2.5 mm / second, for example about 0.2 mm / second. When each whole blood sample with the mixed agglutination reagent moves distally within its respective analysis channel, the plasma moves at a higher speed than the red blood cells. Referring to FIG. 15, each whole blood sample is separated into a red blood cell portion 761 and a plasma portion 763 having a distal liquid-gas interface 765. The red blood cell portion 761 and the plasma portion 763 are connected by a liquid-liquid interface 767. The actuator continues the movement of the red blood cell portion 761 and the plasma portion 763 until the plasma portion 763 fills the second reagent zone 750 and the distal liquid-gas interface 765 contacts the second filling electrode 752, and at the moment when it contacts the second filling electrode 752, the actuator stops the movement. The distal liquid-gas interface 765 of the plasma portion 763 is separated from the surrounding atmosphere surrounding the strip 710 by at least the plasma portion 763 and the red blood cell portion 761.

[0230] Each plasma portion within each second reagent zone aggregates and binds to the first anti-CRP Fab reagent and the second anti-CRP Fab reagent disposed therein. For example, after a short incubation, such as between about 5 - 20 seconds, the actuator begins to oscillate the pressure of each gas at the distal liquid-gas interface within each analysis channel, causing the erythrocyte portion 761 and the plasma portion 763 to oscillate proximally and distally within each channel. The distal liquid-gas interface 767 of each plasma portion is oscillated over a distance near 1 / 2 the length of the second reagent zone, e.g., about ±2 mm, and through a volume near 1 / 2 the volume of the second reagent zone, e.g., ± about 0.325 μL. The cycle time for each complete oscillation is between about 1 second and 5 seconds, e.g., about 2 seconds per oscillation. The oscillation frequency is between about 2 and 10, e.g., about 3. During incubation and oscillation, the plasma portion aggregates the first anti-CRP Fab reagent and the second anti-CRP Fab reagent disposed within each second reagent zone 752.

[0231] Upon completion of incubation and oscillation within each second reagent zone, the actuator then begins to reduce the gas pressure of each distal liquid-gas interface 767, moving the erythrocyte portion and the plasma portion distally within each analysis channel toward their respective gas bladders 760 until the distal liquid-gas interface of each plasma portion contacts the third filling electrode 756 within each analysis channel. The reader activates the magnetic field generator, the optical detector, and the flow actuator, as described for the strip 10 and the reader 111, to capture the magnetic particle reagent within each detection zone, remove plasma containing unbound detectable label, and measure the amount of detectable label retained within the detection zone.

[0232] The various embodiments disclosed herein are exemplary and can be modified. In some embodiments, for example, the microfluidic strip has different forms and / or structures. The microfluidic strip can be formed with fewer than 3 or more layers (e.g., substrates). For example, the strip can be formed by two layers that are fixed together, e.g., adhered, and a microfluidic channel network can be formed on the inner surface of one or both layers (e.g., by stamping, etching, or laser ablation). As another example, the microfluidic strip can be formed by more than 3 layers, a microfluidic channel network or a part thereof can be disposed between each of the plurality of opposing layers, and the connection between the layers can pass through 1 or more layers. The microfluidic strip can be formed of a polymer other than polyester. Suitable polymers include, for example, polydimethylsiloxane (PDMS) elastomers and thermoplastic resins. The microfluidic strip can be formed of a non-polymeric material or a plurality of layers of different materials. For example, one or more rigid layers can be formed from a polymer, quartz, or silicon, and one or more flexible layers can be formed from, for example, a polymer.

[0233] In some embodiments, for example, using optical detection, one or more layers of the strip located above and / or below the detection zone may exhibit high light transmittance in the wavelength range of light irradiation (e.g., fluorescence excitation) into the detection zone and / or in the wavelength range of light emission (e.g., fluorescence emission, scattering, or transmitted irradiation light) from the sample within the detection zone. In some embodiments, fluorescence is excited by excitation light that passes through one layer (e.g., the upper layer) of the strip and reaches the detection zone, and the fluorescence emitted from the detection zone is collected after passing through one layer (e.g., the same layer through which the excitation light passed). The strip may include a non-absorbing layer such that the excitation light and the emitted light pass through the layer facing it. The layer may be arranged, for example, to be located below the layer defining the floor or top of the microchannel of the strip. Alternatively, the surface of the non-absorbing layer may define the floor or top of the channel inside at least a part, for example all, of the detection zone. Non-absorbing means that the layer has a low absorbance for light within at least the range of light emitted by the sample. For example, for fluorescence emission in the visible spectrum, the strip may include a layer with a substantially white appearance when illuminated with substantially colorless light (e.g., sunlight). The non-absorbing layer may have a surface roughness of substantially the same dimensions as the wavelength of the emitted light (e.g., between about 200 nm and about 2500 nm), and the surface may not have a mirror-like finish but may be matte or roughened.

[0234] The layers of the microfluidic strip can also be fixed to each other by techniques other than the adhesive layer. Multiple layers can be fixed to each other by other indirect bonding techniques that use additional materials for fixing the multiple layers, such as, for example, epoxy, adhesive tape, or other chemical reagents. Thermoplastic bonding can be performed with different methods such as adhesive bonding or microwave bonding using an intermediate layer such as a metal or a chemical reagent. As another example, multiple layers can be fixed to each other without the use of, or with minimal use of, additional materials added at the interface between the multiple layers by direct bonding techniques including thermal fusion bonding, ultrasonic welding, surface modification, and solvent bonding. Further examples include anodic bonding, polymer substrate bonding, low-temperature bonding, or high-temperature bonding.

[0235] The microfluidic strip can have a microfluidic channel network different from the microfluidic channel networks of the microfluidic channels 18, 218, 518 or strip 610. For example, the microfluidic channel network can include fewer or more channels or reagent zones and / or detection zones than those described for the microfluidic channel networks of the channels 18, 218, 518 or strip 610. The dimensions of the microfluidic channel network, such as the dimensions of the various channels, reagent zones, detection zones and / or gas bladders, may be different from the microfluidic channel networks of the microfluidic channels 18, 218, 518 or strip 610. The dimensions of the microfluidic network including the channels typically allow capillary action of the sample liquid to flow therein and typically have a volume on the order of pL to μL, for example a volume between about 3 μL and 10 μL. The reagents may differ from those described for the first and second reagent zones and the detection zone of strips 10, 210, 510, 610. In some embodiments, the hematocrit determination channel is arranged in series with the analysis channel rather than being arranged in a separate channel as described for strips 10, 210, 510. Typically, such a series hematocrit determination channel is arranged proximal to the analysis channel such that blood passes through the hematocrit detection zone before reaching the reagent zone of the analysis channel. The sample application zone of the microfluidic strip, such as a port, may include a filter or membrane configured to exclude a portion of the applied sample liquid from entering the microfluidic network of the microfluidic strip. For example, the filter or membrane may be a plasma separation membrane configured to permit plasma to enter the microfluidic network upon application of blood thereto.

[0236] The side cavities of the microchannels of the microfluidic strip, such as the analysis channel, typically have longitudinal axes oriented at a non-zero angle with respect to the longitudinal axis of the microchannel at the location of the opening of the side cavity to the microchannel. For example, each of one or more side cavities of the microchannel may have a longitudinal axis having an angle of at least about 20°, at least about 35°, at least about 45°, at least about 67.5°, or at least about 85°, with respect to the longitudinal axis of the microchannel at the location of the opening of the side cavity to the microchannel. Each of one or more side cavities of the microchannel may have a longitudinal axis having an angle of about 160° or less, about 145° or less, about 135° or less, or about 120° or less, with respect to the longitudinal axis of the microchannel at the location of the opening of the side cavity to the microchannel. For example, the longitudinal axis of each of the plurality of side cavities and the longitudinal axis of the microchannel at the location of such side cavity may be substantially perpendicular to each other.

[0237] The side cavities of the microchannel can be configured and arranged such that the net effect of oscillating the gas pressure at the gas-liquid interface, as disclosed herein, for example, the net effect of oscillating the gas pressure at acoustic frequencies, induces little to no tendency to propel the liquid along the longitudinal axis of the capillary channel, for example, essentially no tendency. In some embodiments, the net effect of the oscillations of the plurality of side cavities is insufficient to propel the liquid at a velocity along the longitudinal axis of the capillary channel that is greater than about 125 μm / s, greater than about 62.5 μm / s, greater than about 30 μm / s, greater than about 25 μm / s, greater than about 15 μm / s, greater than about 7.5 μm / s, or greater than about 0 μm / s. For example, when exposed to oscillations as described herein, the net effect of the plurality of side cavities disposed within the reagent zone or detection zone mobilizes (aggregates) the dry reagent present therein, mixes the sample liquid with the reagent disposed therein, and / or incubates the reaction between the target and the reagent disposed therein, and may induce only insufficient force to propel (expel) the liquid out of such a reagent zone or detection zone for a sufficient period of time. In some embodiments, the longitudinal axis of each of the first set of side cavities within the reagent zone or detection zone can be oriented at a first angle with respect to the longitudinal axis of the microchannel within the reagent zone or detection zone, and the longitudinal axis of each of the second set of side cavities within the reagent zone or detection zone can be oriented at a second angle with respect to the longitudinal axis of the microchannel within the reagent zone or detection zone. Here, the first angle and the second angle are opposite to each other. For example, the opening of each of the first set of side cavities can be oriented generally proximally within the microchannel, and the opening of each of the second set of side cavities can be oriented generally distally within the microchannel. Alternatively, or in combination, the longitudinal axis of each of the plurality of side cavities and the longitudinal axis of the microchannel at the location of such side cavities within the reagent zone or detection zone can be substantially perpendicular to each other.In such an embodiment, the bulk movement of the liquid along the longitudinal axis of the capillary can be induced, for example, by increasing or reducing the gas pressure adjacent to the distal liquid-gas interface of the liquid. This step can be performed sequentially and / or simultaneously with respect to the oscillation of the gas pressure.

[0238] Unlike the strips 10, 210, 510, 610 or the strip of the analysis channel 326, the microfluidic strip can have different arrangements of elements such as, for example, reagents, reagent deposition boundaries, vents, capillary stops, leads, electrodes, and / or bridge contacts, etc. For example, some or all of the elements described as being on the bottom surface can instead be arranged on the top surface or sidewall of the microfluidic channel network, and some or all of the elements described as being on the top surface can instead be arranged on the bottom surface or sidewall of the microfluidic channel network.

[0239] The microfluidic channel networks 18, 218, 518 and the microfluidic network of the strip 610 communicate with the ambient atmosphere 38 via the sample application zones 20, 220, 520, 620 (ports 36, 236, 536, 666). Other configurations are possible. For example, a cap can be attached to the sample introduction zone (port) of the microfluidic channel network that has a sufficient volume, or is configured with a variable volume, to allow the sample liquid to flow and / or move within the microfluidic channel network without inhibition by the generation or reduction of the gas pressure proximal to the sample liquid.

[0240] The microfluidic strip may include a plurality of analysis channels, for example, a plurality of analysis channels configured as analysis channel 26, analysis channel 226, analysis channel 326, analysis channel 526a, 526b, 526c, 526d, or analysis channel 626a, 626b, 626c, 626d, each of which is connected to a common branch channel. Each analysis channel may have its own gas bladder, each of which is operable independently of other gas bladders, allowing independent control over the manipulation of the liquid (e.g., mixing by vibration and / or flow) within the corresponding analysis channel. The reader may be configured with a plurality of flow controllers, such as a flow controller configured as a flow controller of reader 111, including actuators configured to independently control the volume and / or vibration of the corresponding gas bladder, such as piezoelectric actuators like piezoelectric benders. In use, each of the one or more actuators may vibrate in a different phase (e.g., in antiphase) from the vibration of one or more other actuators of the reader. For example, when one or more first actuators compress the respective gas bladders of one or more first analysis channels of the microfluidic strip during a vibration cycle, one or more second actuators simultaneously retreat from (allowing their expansion) the respective gas bladders of one or more second analysis channels of the microfluidic strip during the vibration cycle. Thus, when the first actuator increases the gas pressure distal to the liquid-gas interface of the sample liquid present in one or more first analysis channels, the second actuator reduces the gas pressure distal to the liquid-gas interface of the sample liquid present in one or more second analysis channels. The out-of-phase vibrations may reduce the sound emitted by the system and may result in a quieter operation.

[0241] Each analysis channel of the microfluidic strip can have a function different from that of other analysis channels of the microfluidic strip, for example, the determination of different targets or sample characteristics. A plurality of target or sample characteristics can be determined within a single analysis channel. A single source electrode can be utilized to introduce an electrical signal into the microfluidic channel network, and the signal is detected by the filling electrodes in each of a plurality of different analysis channels. Exemplary microfluidic strip and channel configurations are disclosed, for example, in the aforementioned "Application No. 946".

[0242] The actuator can provide gas pulses in a manner different from that of the actuator of the reader 111. For example, the actuator can apply gas pulses by compressing the lower wall of the microfluidic strip instead of or in addition to the upper wall of the microfluidic strip. The actuator can utilize a vibrating piston or membrane that is in gas communication with the liquid-gas interface of the sample liquid. The reader and the strip can be configured such that a part of the microfluidic channel network of the strip is placed in gas communication with the gas in the reader to apply gas pressure and / or vibration to the liquid-gas interface of the liquid within the microfluidic channel network of the strip. The strip can be configured to apply gas pressure and / or vibration to the proximal gas-liquid interface or the lateral gas-liquid interface adjacent to the sidewall of the channel.

[0243] The microfluidic strip can be configured to allow the introduction of one or more additional liquids other than the sample liquid containing the target. For example, the microfluidic strip can be configured to allow the introduction of a reagent liquid such as a buffer solution through the same sample introduction zone used to introduce the sample liquid or through a separate liquid introduction zone. Alternatively, or in combination, the sample strip can be configured and manufactured to contain a liquid reagent. The liquid reagent can be housed within the hermetically sealed chamber of the microfluidic strip.

[0244] Time T oscThe implementation of the vibration may be different from that described for the operation of the diagnostic system 101. For example, the vibration may not occur during the time T when the liquid is flowing within a particular portion (e.g., reagent zone) of the microfluidic channel network 18, or may occur only during a portion of the time T. The frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be changed during the time T of a particular vibration sequence. The frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be smaller or larger compared to the frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration described for the diagnostic system 101. For example, the frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be implemented as a function of the rate of change of the gas pressure used to move the liquid within the microfluidic channel network. For example, a lower frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration compared to that described for the diagnostic system 101 may be used during the propulsion from the detection zone of the sample liquid and may reduce the likelihood of accidental expulsion of the binding target. As another example, the distance (peak-to-peak) moved by the gas bladder wall during the vibration of the gas bladder wall and / or the actuating member (e.g., actuating foot) driving the vibration of the gas bladder wall during the time T may be at least about 7.5 μm, at least about 12.5 μm, or at least about 15 μm. The peak-to-peak displacement during the vibration of the gas bladder wall and / or the actuating member (e.g., actuating foot) driving the vibration of the gas bladder wall during the time T may be about 60 μm or less, about 50 μm or less, about 40 μm or less, about 17.5 μm or less, about 15 μm or less, about 12.5 μm or less, or about 10 μm or less. mov during which may not occur, or may occur only during a portion of the time T mov The frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be changed during the time T of a particular vibration sequence. osc The frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be smaller or larger compared to the frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration described for the diagnostic system 101. For example, the frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration may be implemented as a function of the rate of change of the gas pressure used to move the liquid within the microfluidic channel network. For example, a lower frequency and / or peak-to-peak displacement of the gas bladder wall induced by the vibration compared to that described for the diagnostic system 101 may be used during the propulsion from the detection zone of the sample liquid and may reduce the likelihood of accidental expulsion of the binding target. As another example, during the time T osc the distance (peak-to-peak) moved by the gas bladder wall during the vibration of the gas bladder wall and / or the actuating member (e.g., actuating foot) driving the vibration of the gas bladder wall may be at least about 7.5 μm, at least about 12.5 μm, or at least about 15 μm. During the time T osc the peak-to-peak displacement during the vibration of the gas bladder wall and / or the actuating member (e.g., actuating foot) driving the vibration of the gas bladder wall may be about 60 μm or less, about 50 μm or less, about 40 μm or less, about 17.5 μm or less, about 15 μm or less, about 12.5 μm or less, or about 10 μm or less.

[0245] Vibration can be performed by vibrating at least a part of the gas bladder at a frequency that is the resonance frequency ωr of the wall of the gas bladder or a frequency substantially the same as this. The resonance frequency ωr of the gas bladder wall can vary as a function of, for example, the tension of the wall of the gas bladder and / or the composition and structure of the wall. For example, the vibration frequency can increase with an increase in the tension of the gas bladder wall and can decrease with a reduction in the tension of the gas bladder wall. By using an actuator such as a piezoelectric actuator (e.g., a piezoelectric bender), the gas bladder wall is vibrated at a frequency ω1, and then, by stopping driving the vibration of the wall at the frequency ω1, the resonance frequency ωr of the gas bladder wall can be determined. When the wall is no longer driven by the actuator, the wall under tension continues to move with a magnitude of movement related to the efficiency of the vibration driven by the actuator at the frequency ω1. The magnitude of the movement can be determined, for example, by using a displacement transducer that converts the movement of the wall into an electrical signal. The displacement transducer can be the actuator used to vibrate the wall at the frequency ω1, and its operating mode can be reversed from the operating mode of the actuator to the operating mode of the displacement transducer. When determining the magnitude of the movement of the wall in response to the wall being vibrated at the frequency ω1, the system uses the actuator again, but at a different frequency ω2, to vibrate the wall. For example, the system can reverse the operation of the displacement transducer and operate it again as an actuator. Next, the system repeats the steps of stopping driving the vibration of the wall, determining the magnitude of the vibration, and vibrating the wall at different frequencies. The magnitude determined is maximum when the vibration frequency corresponds to the resonance frequency ωr. When the resonance frequency ωr is determined, the system continues to drive the vibration of the wall at the resonance frequency ωr or at a frequency substantially the same as it. To ensure that the vibration stays at or near the frequency ωr, the system can perform the steps of stopping driving the vibration of the wall at the frequency ωr or a frequency close to it after driving several cycles of vibration at the frequency ωr or a frequency close to it, determining the magnitude of the vibration, and vibrating the wall at a different frequency ωr'.Here, ωr’ is a frequency in the vicinity of the frequency ωr (for example, within about 3% to 10%). Depending on whether the determined magnitude of the wall vibration is greater or smaller than the vibration at the frequency ωr, the system may continue with the steps of stopping driving the wall vibration, determining the magnitude of the vibration, and vibrating the wall at a different frequency, and may maintain the vibration at a frequency that is the resonance frequency of the wall or a frequency approximately the same as it. For example, the steps of stopping, determining, and (re-)driving the wall vibration (driving) may be repeated at least once for every Nth vibration. Here, N is about 500 or less, about 250 or less, about 125 or less, or about 75 or less. Alternatively, or in combination, the reader may use a non-contact technique such as an optical technique or an acoustic technique to determine the magnitude of the movement of the wall of the gas bladder.

[0246] Time T mov The implementation of the movement of the liquid during may be different from that described for the operation of the diagnostic system 101. For example, the velocity of the liquid may be varied during time T. mov As a particular example, during the step of withdrawing the sample liquid from the detection zone or the reagent zone while retaining a particular material (such as a bound target) within the detection zone or the reagent zone, the sample liquid may be propelled at a first reduced velocity until the sample liquid withdraws from the detection zone or the reagent zone, and then may be propelled at a second higher velocity to facilitate the preparation of the strip for subsequent liquid handling or detection steps. As an alternative to, or in combination with, using gas pressure to induce bulk movement of a liquid or a substance, other techniques such as electroosmosis or other electrokinetic techniques may be used.

[0247] As previously described with respect to strip 10 and system 101, the movement of the sample liquid induced by the vertical retraction and vibration of the actuating end 121 of the piezoelectric bender 117 continues until the distal liquid-gas interface 98 of the sample liquid reaches the third filling electrode 56 at the distal end of the detection zone 54. In some embodiments, the sample liquid is moved a greater distance beyond the detection zone of the strip (or other zone containing a reagent therein), such that the binding reagent disposed within the detection zone (or other zone containing a reagent therein) is exposed to a volume of sample liquid that is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 5 times, or at least about 7.5 times greater than the volume of the detection zone (or other zone containing a reagent therein). In some embodiments, the length of the channel intervening between the detection zone and the gas bladder is increased as compared to the embodiment of strip 10. The filling electrode disposed within the distal portion of such a longer intervening channel can be used to sense the position of the liquid-gas interface of the sample liquid, as previously described. Alternatively or additionally to such a longer intervening channel, the sample liquid can be drawn into the gas bladder, and the volume of the gas bladder can be used to increase the volume of sample liquid that is moved through the detection zone (or other zone containing a reagent therein). The sample liquid that is moved distally into and through the detection zone (or other zone containing a reagent therein) can be moved proximally back into and through the detection zone, as previously described with respect to analysis channel 326 and FIGS. 10 and 11. This process can be repeated a plurality of times, for example, at least 2 times, at least about 3 times, at least about 5 times, or at least about 10 times, thereby increasing the number of opportunities for the binding reagent disposed within the detection zone (or other zone containing a reagent therein) to encounter and bind to a target within the sample liquid. During the time that the sample liquid is being moved (either distally or proximally), a magnetic field generator (such as those previously described) can be used to hold a magnetic binding reagent within the detection zone (or other zone containing a reagent therein).During the sequence of movement of the sample liquid into, through, back into, and through the detection zone (or other zone containing the reagent therein), the movement of the sample liquid can be temporarily stopped to allow incubation with the target present within the same sample volume of the binding reagent therein. During such incubation time, the magnetic field (if used) applied to the zone can be blocked or moved to a position such that it does not exert sufficient force to hold the magnetic particles within the zone. Thereby, the magnetic binding reagent particles can diffuse more freely, allowing more encounters with the target present with the magnetic binding reagent, and allowing accumulation of a greater number of target molecules on the magnetic binding reagent. When the incubation time is completed, the magnetic field is applied again to hold the magnetic particles when the sample liquid is moved, concentrating the magnetic particles within the detection zone. Exemplary incubation times can be, for example, at least about 0.5 minute, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 5 minutes, at least about 10 minutes, or at least about 12 minutes. Exemplary incubation times can be about 15 minutes or less, about 11 minutes or less, or about 7.5 minutes or less. This incubation process can be repeated multiple times, for example, at least 2 times, at least about 3 times, at least about 5 times, or at least about 10 times.

[0248] Diagnostic system 101 uses optical fluorescence to determine the presence of the target, but other techniques, such as other optical techniques like absorption (absorbance) or colorimetry, may be used, and non-optical techniques such as electrochemical techniques can also be used. Strips 10, 210, 510, 610, 610 use immunological techniques, but non-immunological techniques such as enzymological techniques can also be used. Sample liquids other than blood can also be used, which can include, for example, other body fluids such as urine and saliva, and body fluids mixed with other reagents and liquids such as anticoagulants or buffers.

[0249] Exemplary suitable technologies, targets, and sample liquids are disclosed, for example, in the aforementioned "Application No. 946". Exemplary targets include viral, fungal, or bacterial pathogens such as influenza, coronavirus (e.g., SARS-CoV-2), MRSA, c.diff (Clostridium difficile), flavivirus, Candida, cryptococcus, etc., and also include antibodies against antigens from such pathogens. Exemplary reagents and methods for determining coronavirus-related targets are included in U.S. Provisional Patent Application No. 62 / 992,681 filed on March 20, 2020, U.S. Provisional Patent Application No. 63 / 009,906 filed on April 14, 2020, and U.S. Provisional Patent Application No. 63 / 032,378 filed on May 29, 2020. Each of these is titled "Coronavirus Assay" and is hereby incorporated by reference in its entirety. Exemplary reagents and methods for determining pathogens such as virus-related targets like coronavirus and dengue fever-related targets are disclosed in UK Patent Application No. 2006306.1 titled "Infectious Disease Assay" filed on April 29, 2020. The entire application is hereby incorporated by reference in its entirety. The reagents and methods as disclosed in the aforementioned applications can be used or implemented in combination with the strips, readers, systems, and methods disclosed herein.

[0250] In some embodiments, the strip includes a lysis reagent containing a sufficient amount of exonuclease to release viral proteins (e.g., nucleocapsid protein) from viral RNA. The release of protein from RNA increases the amount of protein available for use in reactions (e.g., immunological reactions) to determine the presence of proteins in the sample. Exemplary protein targets include the nucleoproteins (e.g., nucleocapsid) of HIV and coronavirus (e.g., SARS-CoV-2). An exemplary exonuclease is Benzonase® nuclease.

[0251] In some embodiments, lysis can be performed in the presence of a salt concentration of at least about 0.2 M, at least about 0.3 M, or at least about 0.4 M. The salt concentration can be about 1.2 M or less, about 1.1 M or less, about 1.0 M or less, or about 0.9 M or less. Exemplary salts include chloride salts such as sodium chloride or potassium chloride, and combinations thereof.

[0252] In some embodiments, the strip includes an integrity monitoring reagent configured to determine whether the strip has been exposed to ambient atmosphere or humidity conditions, which indicates a failure of the hermetically sealed pouch and / or exposure of the sealed pouch to excessive temperature. Typically, the integrity monitoring reagent is disposed within the strip in a manner similar to the microfluidic channel network, but in a separate channel or chamber separated from the microfluidic channel network so as not to contaminate the sample liquid or analytical reagent. The channel or chamber has a vent or other opening for exposing the integrity monitoring reagent to the gas within the pouch. The reader is configured to monitor the integrity monitoring reagent using fluorescence or colorimetry to determine a change indicating a harmful environmental condition or poor airtightness of the pouch.

[0253] [Examples] The following examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0254] [Example 1: SARS-CoV-2 Ab Assay] The diagnostic system disclosed herein, including a test strip and a reader, It was used to perform a SARS-CoV-2 Ab immunofluorescence assay for the qualitative detection of total antibodies against SARS-CoV-2 in a blood-based sample liquid, such as whole blood (capillary finger stick or venous), plasma, or serum. The SARS-CoV-2 Ab assay is intended to be used to help identify (detect) individuals who have an adaptive immune response against SARS-CoV-2 Abs indicating recent or previous infection. The result is the detection of SARS-CoV-2 antibodies.

[0255] Referring to FIG. 16, the SARS-CoV-2 Ab strip has, proceeding upward from the lower left, a sample application zone, a tapered common supply channel, a branched channel, and, along the branched channel from right to left in the drawing, four analysis channels and one hematocrit channel, the proximal portions of which define a microfluidic channel network including an excitation electrode (also called a supply electrode) and a common electrode. As described below, the common electrode extends across each of the hematocrit channel and the four analysis channels.

[0256] Each of the four analysis channels is configured and arranged to facilitate determination of the presence and / or amount of a target present in the sample liquid. Proceeding distally from the branched channel along the longitudinal axis of each analysis channel, the analysis channel includes a vent, a capillary stop, a common electrode (common electrode), a reagent zone, a first filling electrode, a second filling electrode, a detection zone, a third filling electrode, an interval channel, and a gas bladder.

[0257] In use, a sample is applied to a sample application zone and flows by capillary action along a tapered common supply channel to a branch channel. Along the branch channel, a first portion of the sample liquid flows by capillary action to each of four analysis channels, and a second portion of the sample liquid flows by capillary action to a hematocrit channel. As described, for example, for reader 111 of strip 10 and supply electrode 70, the reader causes an excitation electrode (supply electrode) to generate a time-varying signal. When the strip is properly filled with the sample liquid, the sample liquid establishes conduction (continuity) between the excitation electrode and the common electrode along each of the following five paths: (1) from the portion of the excitation electrode that crosses the proximal portion of the hematocrit channel, along the hematocrit channel, to the portion of the common electrode that crosses the hematocrit channel, and (2)-(5) from the portion of the excitation electrode that crosses the proximal portion of the hematocrit channel, along the branch channel, along the proximal portion of each analysis channel, to each respective portion of the common electrode that crosses such an analysis channel. The reader determines proper filling of the branch channel and the four analysis channels based on the time-varying signal measured at the contact of the common electrode at the periphery of the strip. When conduction (continuity) is established along all five paths, the total impedance between the excitation electrode and the common electrode is minimized compared to that total impedance when conduction (continuity) is not established along one or more paths (e.g., when one or more of the analysis channels are not properly filled). Thus, the common electrode provides the ability to confirm that each of the multiple channels of the strip is properly filled using only two electrodes (the excitation / supply electrode and the common electrode) and only two contacts at the periphery of the strip (each respective contact corresponding to each electrode).

[0258] [General principle of operation of the SARS-CoV-2 Ab assay] The SARS-CoV-2 Ab assay forms a bridging particle-particle sandwich immunoassay that measures antibodies specific for SARS-CoV-2 present in a test sample using a SARS-CoV-2 specific antigen.

[0259] A dry reagent containing SARS-CoV-2 specific antigen-labeled fluorescent particles and SARS-CoV-2 specific antigen-labeled biotin is present in dried form within the first reagent zone of each of the four analysis channels. The sample liquid applied to the strip reconstitutes the dried reagent. The reader uses a piezoelectric actuator to move the sample liquid and mix it with the reagent as described for the diagnostic system 101. If SARS-CoV-2 antibodies are present in the sample liquid, an antigen bridge sandwich complex is formed with the fluorescent particle-labeled SARS-CoV-2 antigen and the biotin-labeled SARS-CoV-2 antigen. After incubation, the resulting immune complex is transferred to the detection zone where the reagent is mixed with streptavidin-labeled magnetic particles that bind to the biotin sandwich complex. A magnetic field is applied to the measurement (detection) zone to attract the magnetic particles and the associated SARS-CoV-2 antibody immune complex. The fluid control system of the reader acting on the strip removes the sample liquid and unbound label from the measurement zone by operating (e.g., compressing) the piezoelectric actuator of the gas bladder at the distal end of each analysis channel. When the sample liquid is removed from the detection zone together with the unbound label, the reader measures the fluorescence signal of the essentially dry immune complex fluorescent particles. It is proportional to the concentration of SARS-CoV-2 antibodies in the sample liquid.

[0260] The reader operates the hematocrit channel to facilitate an optical determination of the hematocrit of the blood-based sample liquid applied to the sample application zone, as described for strip 10, without the reagent.

[0261] [Composition of the strip reagent] Three of the four analysis channels of the SARS-CoV-2 Ab strip are each used to detect antibodies in the sample liquid. The fourth analysis channel contains an on-board control reagent (OBC) that is used to verify proper assay operation. The SARS-CoV-2 assay is constructed using highly specific antigens of the SARS-CoV-2 virus to ensure high specificity and low cross-reactivity. The reagent contains the receptor binding domain (RBD) and spike glycoprotein S1 (S1) of the SARS-CoV-2 virus.

[0262] SARS-CoV-2 (2019-nCoV) spike S1-His was obtained from Sino Biological Inc. (Catalog number 40591-V08H, Beijing, China). This protein was constructed by expressing a DNA sequence encoding the SARS-CoV-2 (2019-nCoV) spike protein S1 subunit (YP_009724390.1) (Val16-Arg685) with a polyhistidine tag attached to the C-terminus. The spike S1-His was then conjugated to biotin (A39259, Thermo Fisher Scientific, Waltham, Massachusetts) or fluorescent latex particles.

[0263] The SARS-CoV-2 (2019-nCoV) spike RBD-mFc was obtained from Sino Biological Inc. (Catalog No. 40592-V05H, Beijing, China). This protein was constructed by expressing a DNA sequence encoding the SARS-CoV-2 (2019-nCoV) spike protein RBD (YP_009724390.1) (Arg319-Phe541) with the Fc region of mouse IgG1 attached to the C-terminus. The spike RBD-Fc was then conjugated to biotin (A39259, Thermo Fisher Scientific, Waltham, Massachusetts) or fluorescent latex particles.

[0264] The composition of the 4-channel strip assay was as follows. · Analytical channel 1 S1-S1 bridge serological assay: SARS-CoV-2 S1 spike glycoprotein-biotin conjugate SARS-CoV-2 S1 spike glycoprotein-latex conjugate · Analytical channel 2 RBD-S1 bridge serological assay: SARS-CoV-2 S1 spike glycoprotein-biotin conjugate SARS-CoV-2 receptor binding domain RBD-latex conjugate · Analytical channel 3 RBD-S1 bridge serological assay: SARS-CoV-2 S1 spike glycoprotein-biotin conjugate SARS-CoV-2 receptor binding domain RBD-latex conjugate · Analytical channel 4 OBC on-board control: Biotinylated-latex conjugate Streptavidin-magnetic particle conjugate

[0265] The components and immune complex formation of the S1-S1 bridge serological assay and the RBD-S1 bridge serological assay are illustrated in FIGS. 17A and 17B, respectively. FIG. 17A shows the S1-S1 bridge immunoassay, and FIG. 17B shows the RBD-S1 bridge immunoassay. The onboard control assay is illustrated in FIG. 18.

[0266] [Operation of Reader and Strip] The user selects SARS-CoV-2 from the menu of the assay reader. The reader performs a self-check to verify that the power system, electronic system, electromechanical system, and software system are operating correctly. The user inserts the strip into the reader and applies the sample liquid to the sample application zone of the strip. The sample liquid is a blood-based sample liquid such as whole blood (e.g., finger stick or venous), plasma, or serum. The reader operates the strip to perform the assay as described for the strips of diagnostic system 101, strips 10, 210, 510, or analysis channel 326.

[0267] [Analytical Performance of Assay] (Sensitivity and Specificity of Analysis) Reactivity / Inclusivity: Mutations in the SARS-CoV-2 genome have been identified as the virus spreads, but the inventor has not, at present, recognized serologically unique strains described compared to the originally isolated virus.

[0268] Cross-reactivity: The SARS-CoV-2 Ab test did not cross-react positively with the sample liquid for antibodies against hepatitis C virus, hepatitis B virus (genotype D), or HIV; human coronaviruses (HKU1, NL63, OC43, and 229E), antinuclear antibodies, antigen influenza A, influenza B, respiratory syncytial virus; heterophil (heterophilic) antibodies against mononucleosis. The results are shown in Table 1. Table 1: Cross-reactivity of the SARS-CoV-2 Ab test TIFF0007716412000001.tif78144

[0269] [Agreement rate in the clinical trial]

[0270] i) Positive agreement rate (Symptomatic subjects in the endemic (local epidemic)) The positive agreement rate was evaluated using plasma samples collected from symptomatic subjects (see Table 2). All subjects were confirmed to be positive for the novel coronavirus 2019 by RT-PCR. The positive population consisted of the following subjects. · 22 who lived in the UK during the COVID-19 pandemic in 2020 · 52 who lived in the US during the COVID-19 pandemic in 2020 Table 2: Positive agreement rate of the SARS-CoV-2 Ab test by days after PCR: Symptomatic subjects in the endemic TIFF0007716412000002.tif52132

[0271] ii) Negative agreement rate (Symptomatic subjects in the endemic (local epidemic)) Using 15 samples (EDTA plasma samples) collected from symptomatic subjects living in the UK, the negative agreement rate of the SARS-CoV-2 Ab test was evaluated and is shown in Table 3. The samples were collected during the COVID-19 pandemic in 2020 and all were confirmed to be negative for the novel coronavirus 2019 by RT-PCR. Table 3: Negative agreement rate of the SARS-CoV-2 Ab test: Symptomatic subjects in the endemic TIFF0007716412000003.tif26129

[0272] (Asymptomatic subjects with endemic (local epidemic)) Furthermore, the negative agreement rate of the SARS-CoV-2 Ab test was evaluated using 22 presumptive negative plasma specimens collected from asymptomatic subjects in the UK during the 2020 COVID-19 pandemic. Compared with the expected results of all asymptomatic subjects with endemic, the negative agreement rate obtained as the result of the SARS-CoV-2 Ab test was 100% (22 / 22 = 100%). The results are shown in Table 4 below. Table 4: Negative agreement rate of the SARS-CoV-2 Ab test: Presumptive negative asymptomatic subjects with endemic TIFF0007716412000004.tif31145

[0273] (Asymptomatic subjects without endemic) Furthermore, the specificity of the SARS-CoV-2 Ab test was evaluated using 262 presumptive negative plasma specimens collected from asymptomatic subjects before the COVID-19 pandemic. 33 samples from asymptomatic subjects in the United States collected in 2016 were supplied trademarkly from the Biotechnology Research Service, 66 samples collected in 2019 in the United States before the COVID-19 pandemic were supplied commercially from blood donation centers, and 163 samples from asymptomatic subjects in the UK were collected during the prior clinical trial evaluation under an approved protocol before the COVID-19 pandemic. All samples were collected between 2016 and October 2019. Compared with the expected results, the negative agreement rate obtained as the result of the SARS-CoV-2 Ab test was 100% (262 / 262 = 100%), and is shown in Table 5. Table 5: Negative agreement rate of the SARS-CoV-2 Ab test: Asymptomatic subjects without endemic TIFF0007716412000005.tif36138

[0274] (Overall Results) The negative agreement rate obtained as a result of the SARS-CoV-2 Ab test compared with the predicted results was in the 95% confidence interval of 98.8 - 100%, and was 100% (299 / 299 = 100%).

[0275] [Example 2: SARS-CoV-2 Ag Assay] The diagnostic system disclosed herein, including the test strip and the reader, was used to perform a SARS-CoV-2 Ag assay for the qualitative detection of nucleoprotein antigen against SARS-CoV-2 after a swab was added to either nasal and nasopharyngeal swab specimens or a universal transport medium (UTM) or viral transport medium (VTM) collected from an individual suspected of having COVID-19.

[0276] The result is the identification of the SARS-CoV-2 nucleoprotein antigen. The antigen is generally detectable in nasal and nasopharyngeal swabs during the acute phase of infection.

[0277] Referring to FIG. 19, the strip defines a microfluidic channel network including, proceeding upward from the lower left, a sample application zone, an arcuate common supply channel, a branching channel, four analysis channels from right to left in the drawing, a common electrode, an excitation electrode (supply electrode), and a narrow vent channel terminating in a vent (as described for vent channel 576 and vent 576a of microfluidic strip 510).

[0278] In use, a sample is applied to a sample application zone and flows by capillary action along an arcuate common supply channel to branch channels. Along the branch channels, a first portion of the sample liquid flows by capillary action to each of four analysis channels, and a second portion of the sample liquid flows by capillary action to excitation / supply electrodes and a common electrode and stops moving at the proximal end of a narrow vent channel. As described, for example, in Example 1 and for reader 111 of strip 10 and supply electrode 70, the reader causes the excitation electrode (supply electrode) to generate a time-varying signal. When the strip is properly filled with the sample liquid, the sample liquid establishes conduction (continuity) between the excitation electrode and the common electrode along each of the following five paths: (1) from the leftmost portion where the excitation electrode crosses the branch channel, along the branch channel to the portion where the common electrode crosses the branch channel, and (2)-(5) from the portion where the excitation electrode crosses the branch channel, along the branch channel, along the proximal portion of each analysis channel, to the respective portions where the common electrode crosses such analysis channels. As explained in Example 1, the reader determines proper filling of the branch channel and the four analysis channels based on a time-varying signal measured at the contact of the common electrode at the periphery of the strip.

[0279] [Principle of SARS-CoV-2 Ag Test] The SARS-CoV-2 Ag assay is a point-of-care rapid microfluidic immunoassay. The assay uses SARS-CoV / SARS-CoV-2 specific antibodies in a particle-particle sandwich immunoassay to determine the presence of SARS-CoV-2 nucleocapsid protein (NP) present in a test sample.

[0280] The reader uses piezoelectric actuators to compress / decompress the gas bladder of each analysis channel to provide for the movement of the sample liquid and the mixing of the reagent and the sample liquid within the microchannel network of the strip. A magnetic field is applied to the measurement (detection) zone to capture magnetic particles and the accompanying SARS-CoV-2NP immune complex. Before detecting the complex, the piezoelectric actuator of each channel compresses the corresponding gas bladder to release the sample liquid along with unbound labels from the detection zone. The reader measures the fluorescence signal of the immunocomplex fluorescent particles in an essentially dry state. It is proportional to the concentration of the SARS-CoV-2 virus NP antigen in the sample liquid.

[0281] [Form of the test strip] The SARS-CoV-2 Ag test uses two independent assay channels in the strip to analyze the NP antigen in the test sample (see Figure 5). A third independent assay channel performs a test for IgA in the sample. A fourth assay channel contains a strip on-board control reagent (OBC) that is used to verify that the test has operated properly.

[0282] The configuration of the 4-channel test strip assay was as follows. · Channel 1 RBD-IgA serological assay (reported selectively): SARS-CoV-2 anti-IgA-biotin complex pre-bound to streptavidin-magnetic particles SARS-CoV-2 receptor binding domain RBD-latex complex · Channel 2 NP antigen assay: SARS-CoV / SARS-CoV-2 nucleocapsid antibody, mouse MAb-latex SARS-CoV / SARS-CoV-2 nucleocapsid antibody, rabbit Mab-magnetic particles · Channel 3 NP antigen assay: SARS-CoV / SARS-CoV-2 Nucleocapsid Antibody, Mouse MAb-Latex SARS-CoV / SARS-CoV-2 Nucleocapsid Antibody, Rabbit Mab-Magnetic Particle · Channel 4 OBC On-Board Control: Biotinylated-Latex Complex Pre-Bound to Streptavidin-Magnetic Particles

[0283] The SARS-CoV / SARS-CoV-2 nucleocapsid antibody, mouse Mab, was obtained from Sino Biological Inc. (40143-MN05). The SARS-CoV / SARS-CoV-2 nucleocapsid antibody, rabbit Mab, was obtained from Lumira Dx UK Ltd. (SD-QMS-WI-30066).

[0284] The description of the SARS-CoV-2 Ag nucleocapsid protein immunoassay - Channels 2 and 3 is shown in Figure 20.

[0285] The description of the RBD-IgA serological assay - (Optionally reported) - Channel 1 is shown in Figure 21.

[0286] The description of the on-board control assay - Channel 4 is shown in Figure 22.

[0287] [Operation of Reader and Strip] The preparation and testing of the samples were conducted as follows. The sample liquid was a nasal and / or nasopharyngeal swab specimen, or a swab specimen combined with a universal transport medium (UTM) or a viral transport medium (VTM). The nasal and / or nasopharyngeal swabs were obtained from a single subject and placed in an extraction buffer. The extraction buffer could be held within an extraction container as described in U.S. Provisional Patent Application No. 63 / 029,579, entitled "Extraction Container," filed on May 25, 2020. The entire disclosure of that application is incorporated herein by reference. For the analysis of NP antigen in VTM, the swab was first extracted into VTM, then 700 microliters of VTM was directly added to the extraction buffer container and agitated by rotating the swab 5 times against the side of the vial. The swab was then removed from the extraction vial while squeezing the middle of the extraction container to remove the liquid from the swab. The container was sealed with a dropper cap.

[0288] Referring to FIG. 23, a schematic diagram of the "RBD-IgA serology assay - (optionally reported) - channel 1" illustrates the formation of an initial complex comprising (1) an anti-IgA antibody-biotin conjugate, (2) anti-SARS-CoV-2 IgA present in the sample, and (3) RBD fluorescent latex particles. Next, the initial complex binds to Mag (magnetic particle)-streptavidin (capture reagent), which is held in place by the magnet of the reader for fluorescence detection. In the following examples, the assay was performed using a strip containing, in dry form, (1) an anti-IgA antibody-biotin conjugate (conjugate) pre-bound to a complex of streptavidin and magnetic particles (conjugate), and (2) RBD fluorescent latex particles. When a liquid sample was applied to the strip, a complex as shown on the right side of the arrow was formed. The complex was held in place by the magnet of the reader for fluorescence detection.

[0289] Similarly, in the onboard control assay, the strip, in dry form, as shown in Figure 24, contained (1) a fluorescent latex particle-biotin conjugate that was pre-bound to a complex (conjugate) of streptavidin and magnetic particles.

[0290] The user selects SARS-CoV-2 Ag from the menu of the assay reader. The reader performs a self-check to verify that the power system, electronic system, electromechanical system, and software system are operating correctly. The user inserts the strip into the reader and applies 1 drop of the sample liquid to the sample application zone of the strip using the dropper cap. The reader operates the strip to perform the assay as described for the diagnostic system 101, strip 10, 210, 510, or the strip of the analysis channel 326.

[0291] The "Calibration LCF" file returns the quantitative untransformed final optical signal from each channel on the instrument screen. Channels 1-3 (looking from left to right across the test strip) are the assay channels, while channel 4 is the OBC channel.

[0292] This file defines the four aforementioned assays. All are displayable. Each assay is assigned to 1 calibration curve (calibration graph) and 1 wavelength band as shown in Table 6. Table 6: Assay Summary TIFF0007716412000006.tif73148

[0293] A single calibration curve is defined for each assay for all acceptable sample types. In this file, all of the main channel assay curves and OBC are the same, and a simple 1:1 untransformed calibration table is used in all cases.

[0294] An additional index of displayable results is defined as generating an average using the outputs from Assays 2 and 3.

[0295] Two quality control levels are defined (Index 1 = positive, Index 2 = negative) and applied to Index 1, 2, and 3 of the results. However, in all cases, the limit values are from 0 to 1,000,000.

[0296] [Limit of Detection (LoD) - Analytical Sensitivity] (LoD Study 1) The LoD study determines the lowest detectable concentration of SARS-CoV-2. At this concentration, approximately 95% of all (truly positive) replicate the positive test. The LoD of the aforementioned antigen detection assay was determined by a limit dilution study using a characterized SARS-CoV-2 culture heat-inactivated virus (Zeptometrix Corporation, 0810587CFHI - 0.5ml, Lot324307).

[0297] Severe Acute Respiratory Syndrome-related Coronavirus 2 (isolate: USA-WA1 / 2020) is an enveloped positive-sense single-stranded RNA virus derived from the Coronaviridae family and the Betacoronavirus genus. The stock virus was isolated from a patient with a respiratory disease who developed COVID-19 in January 2020 in Washington, USA, after returning from a trip to an infected area in China. The genomic sequence can be found in GenBank MN985325.

[0298] Each frozen aliquot contains 0.50 mL of the heat-inactivated virus culture. The pre-inactivation titer was determined from the infectious aliquot. The inactivation of the virus was confirmed (verified) after heat inactivation by the absence of virus growth in a tissue culture-based infectious assay. (Zeptometrix product description, www.zeptometrix.com / media / documents / PI0810587CFHI-0.5mL.pdf)

[0299] Serial two-fold dilutions of characterized SARS-CoV-2 aliquots were tested in triplicate. The lowest concentration at which all three replicates were positive was treated as the provisional LoD for each test. The LoD for each test was then confirmed by testing 20 replicates at the concentration of the provisional limit of detection. The final LoD for each test was determined as the lowest concentration that resulted in a positive detection of 19 out of 20 replicates, as shown in Figure 25A.

[0300] The LoD study using heat-inactivated SARS-CoV-2 virus in cell culture (Zeptometrix, 0810587CFHI-0.5ml, lot 324307) shows that the LoD is in the range of 1:6400 dilution to 1:12800 dilution, i.e., 118 - 236 TCID50 / ml (median tissue culture infective dose), as shown in Figure 25B.

[0301] The LoD study using a dilution series of patient nasopharyngeal swab samples (characterized as PCR positive with a CT = 30; CT is the cycle threshold, defined as the number of cycles required for the fluorescence signal to exceed the background level) processed with extraction tubes and buffers for the SRAS-CoV-2 Ag test shows that the LoD is less than 1 at a 256-fold dilution, i.e., Ct value ≤ 38.

[0302] (LoD Study 2) The LoD of the SARS-CoV-2 Ag test was established using serial dilutions of gamma-irradiated SARS-CoV-2 (BEI Resources NR-52287). NR-52287 is a preparation of SARS-related coronavirus 2 (SARS-CoV-2) isolate USA-WA1 / 2020 inactivated by gamma irradiation at 5 × 10 6 RAD. The material was supplied frozen at a concentration of 2.8 × 10 5 TCID50 / mL.

[0303] The study to determine the LoD of the SARS-CoV-2 Ag assay was designed to reflect the assay when using direct nasal swabs. In this study, the starting material was spiked into volumes of pooled human nasal matrix obtained from healthy donors and confirmed to be negative for SARS-CoV-2. At each dilution, 50 μL of the sample was added to the swab and the swab was processed for testing in the SARS-CoV-2 Ag assay using procedures appropriate for patient nasal swab specimens according to the accompanying documentation. The LoD was determined in three steps (in accordance with the CLSI standard, Evaluation of the Detection Capability of Clinical Laboratory Measurement Procedures, CLSI EP17).

[0304] a. Limit of Detection Screening The first LoD screening study was performed using a 5-fold serial dilution (6 dilutions total) of gamma-irradiated virus made in pooled negative human nasal matrix starting at a test concentration of 2×10 4 TCID50 / mL (as shown in Table 7 below) and processed for each study as described above. These dilutions were tested in triplicate. The lowest concentration at which all (3 out of 3 replicates) were positive was selected for LoD range determination. This was 32 TCID50 / mL. Table 7: Analysis of the Limit of Detection of SARS-CoV-2 TIFF0007716412000007.tif52142

[0305] b. Limit of Detection Range Determination Using a concentration of 32 TCID50 / mL, the LoD was further refined using a 2-fold serial dilution (4 dilutions total) of gamma-irradiated SARS-CoV-2 virus made in pooled negative human nasal matrix. These dilutions were tested in triplicate. The lowest concentration at which all (3 out of 3 replicates) were positive was treated as the provisional LoD of the SARS-CoV-2 Ag assay. This was 32 TCID50 / mL. Table 8: Analysis of the Detection Limit of SARS-CoV-2 after Gamma Irradiation TIFF0007716412000008.tif41146

[0306] c. Confirmation of the detection limit Next, the LoD of the SARS-CoV-2 Ag test was confirmed by testing 20 replicates at the concentration of the provisional detection limit. The final LoD of the SARS-CoV-2 Ag test was determined to be the lowest concentration that resulted in 20 positive detections out of 20 replicates. Based on this test, the LoD of the nasal swab specimens was confirmed to be 32 TCID50 / mL. Table 9: Summary of the Confirmation Analysis of the Detection Limit TIFF0007716412000009.tif21151

[0307] [Cross-reactivity (specificity of the analysis)] The cross-reactivity of the SARS-CoV-2 Ag test was evaluated by testing a panel of related pathogens, pathogens with high morbidity, and normal or pathogenic flora that could reasonably be encountered in clinical specimens and potentially cross-react with the SARS-CoV-2 Ag test. They included various microorganisms, viruses, and negative matrices. Each microorganism and virus was tested in the absence or presence of heat-inactivated SARS-CoV-2 at 3×LoD. The final concentrations of the microorganisms and viruses are listed in Table 10 below (for bacteria, a concentration of 10 6 CFU / mL or higher, and for viruses, a concentration of 10 5 pfu / mL or higher is recommended). For some microorganisms, the stock concentration was below the recommended test concentration. In these cases, it was only possible to test these microorganisms at the stock concentration. Table 10: Analysis of the Cross-Reactivity of the Displayed Microorganisms with the SARS-CoV-2 Test TIFF0007716412000010.tif203136

[0308] To estimate the potential cross-reactivity of the SRAS-CoV-2 Ag test in microorganisms that could not be used in the wet test, a computer analysis using the Basic Local Alignment Search Tool (BLAST) maintained by the National Center for Biotechnology Information (NCBI) in the United States was used to evaluate the degree of protein sequence homology.

[0309] For human coronavirus HKU1, there is homology between the SARS-CoV-2 nucleocapsid protein and human coronavirus HKU1. The BLAST results showed 30 sequence IDs indicating homology, all nucleocapsid proteins. It was found that sequence ID AGW27840.1 had the highest alignment score and 39.1% homology over 76% of the sequence. This value is relatively low, but cross-reactivity cannot be completely excluded.

[0310] For SARS-coronavirus, there is high homology between the SARS-CoV-2 nucleocapsid protein and SARS-coronavirus. The BLAST results showed 68 sequence IDs indicating homology, mostly nucleocapsid proteins. It was found that sequence ID AAR87518.1 had the highest alignment score isolated from a human patient and 90.76% homology over 100% of the sequence. This value is high and there is cross-reactivity.

[0311] Regarding the MERS coronavirus, there is a high homology between the SARS-CoV-2 nucleocapsid protein and the MERS coronavirus. The results of BLAST showed at least 114 sequence IDs indicating homology, mostly nucleocapsid proteins. Sequence IDs AHY61344.1 and AWH65950.1 had the highest alignment scores isolated from human patients and were found to have 49.4% and 50.3% homology over 88% of the sequences. This potentially indicates moderate cross-reactivity, but testing of the MERS virus at 7930 PFU / mL showed no reactivity (see the table above).

[0312] [Research on microbial interference] To show that false negatives do not occur when SARS-CoV-2 is present in the sample together with other microorganisms, microbial interference in the SARS-CoV-2 Ag test was evaluated by testing a panel of relevant pathogens, pathogens with high morbidity, and normal flora or pathogenic flora. They include various microorganisms, viruses, and negative matrices. Each microorganism and virus was tested in triplicate in the absence or presence of heat-inactivated SARS-CoV-2 at 3×LoD. The final concentrations of the microorganisms and viruses are listed in Table 11 below (for bacteria, a concentration of 10 6 CFU / mL or higher, an...

Claims

1. A method for detecting at least one target substance in a sample liquid, comprising: (a) introducing the sample liquid into a microfluidic channel of a microfluidic device, wherein the microfluidic channel contains a gas therein, and the sample liquid contacts the gas, thereby forming a sample liquid-gas interface therebetween; (b) moving the sample liquid and the sample liquid-gas interface to a first zone by reducing the pressure of the gas, wherein the first zone contains a first reagent disposed therein; (c) mixing the sample liquid with the first reagent by vibrating the pressure of the gas at an acoustic frequency to form a first mixture. The method is characterized in that: The step of vibrating the pressure of the gas is i) carried out simultaneously with the latter, ii) carried out after the latter, or iii) carried out simultaneously with and after the latter; the acoustic frequency is between 100 Hz and 3000 Hz and does not induce bulk movement of the sample liquid. A method characterized by the above.

2. The step of vibrating the pressure of the gas is carried out at a frequency of at least 500 Hz and not more than 2000 Hz. The method according to claim 1, characterized by the above.

3. The first reagent includes a lysis reagent, a binding reagent, or an optical label. The method according to claim 1, characterized by the above.

4. The step of vibrating the pressure of the gas is carried out after the step of reducing the pressure of the gas. During the step of vibrating the pressure of the gas, the position of the sample liquid-gas interface with respect to the longitudinal axis of the microfluidic channel remains substantially unchanged. The method according to claim 1, characterized by the above.

5. The step of reducing the pressure of the gas includes increasing the internal distance between a first inner wall and a second inner wall of a bladder region located at the distal portion of the microfluidic device. The bladder region, the first inner wall and the second inner wall are in direct contact with the gas, and the sample liquid-gas interface and the microfluidic channel are in gas communication. The method according to claim 1, characterized by the above.

6. The step of vibrating the pressure of the gas includes the step of vibrating the internal interval between the first inner wall and the second inner wall at one or more acoustic frequencies. The method according to claim 5, characterized in that.

7. The step of vibrating the internal interval includes the step of changing the internal interval over a total peak-to-peak distance of 5 μm to 75 μm, The total peak-to-peak distance is measured along an axis perpendicular to the plane defined by the microfluidic device. The method according to claim 6, characterized in that.

8. (a) A step of moving the first mixture and the sample liquid-gas interface from the first zone to the second zone by reducing the pressure of the gas again, wherein the second zone contains a second reagent disposed therein. And, (b) A step of mixing the first mixture with the second reagent by vibrating the pressure of the gas again at an acoustic frequency to form a second mixture. further comprising The step of vibrating the pressure of the gas again is relative to the step of reducing the pressure of the gas again. i) being carried out simultaneously with the latter, ii) being carried out after the latter, or iii) being carried out simultaneously with and after the latter, The acoustic frequency is between 100 Hz and 3000 Hz and does not induce bulk movement of the sample liquid. The method according to claim 6, characterized in that.

9. (a) A step of moving the second mixture and the sample liquid-gas interface from the second zone to the third zone by further reducing the pressure of the gas, wherein the third zone contains a third reagent disposed therein. And, (b) A step of mixing the second mixture with the third reagent by vibrating the pressure of the gas again at an acoustic frequency to form a third mixture. further comprising The step of vibrating the pressure of the gas further again is relative to the step of reducing the pressure of the gas further again. i) being carried out simultaneously with the latter, ii) being carried out after the latter, or iii) being carried out simultaneously with and after the latter, The acoustic frequency is between 100 Hz and 3000 Hz and does not induce bulk movement of the sample liquid. The method according to claim 8, characterized in that.

10. Activating a magnetic field generator to move the target substance towards the inner wall of the microfluidic channel further comprising wherein the target substance is bound to magnetic particles The method according to claim 9, characterized in that

11. (a) By increasing the pressure of the gas, the third mixture not bound to the magnetic particles and the sample liquid-gas interface are moved from the third zone to i) the second zone, ii) the first zone, iii) the capillary stop, or iv) the sample application port, wherein the target substance bound to the magnetic particles remains within the third zone; (b) simultaneously with increasing the pressure of the gas, vibrating the pressure of the gas at an acoustic frequency further comprising wherein the acoustic frequency is between 100 Hz and 3000 Hz and does not induce bulk movement of the sample liquid The method according to claim 10, characterized in that

12. (a) irradiating the third zone with light to thereby emit a signal to an optical label; (b) detecting the signal via an optical detector to thereby indicate the presence of the target substance in the sample liquid The method according to claim 11, further comprising

13. The step of vibrating the pressure of the gas again, the step of vibrating the pressure of the gas still further, or the step of vibrating the pressure of the gas when increasing the pressure of the gas includes vibrating the corresponding internal distance between the first inner wall and the second inner wall The method according to claim 12, characterized in that

14. The step of vibrating the corresponding internal distance includes changing the corresponding internal distance over a full peak-to-peak distance of 75 μm or less, 65 μm or less, 50 μm or less, 40 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, 10 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, at least 1 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 10 μm, at least 15 μm, or at least 20 μm, wherein the full peak-to-peak distance is measured along an axis perpendicular to the plane defined by the microfluidic device The method according to claim 13, characterized in that

15. The step of vibrating the pressure of the gas again, the step of vibrating the pressure of the gas still again, or the step of vibrating the pressure of the gas when increasing the pressure of the gas is performed at a frequency of at least 500 Hz, at a frequency of at least 700 Hz, at a frequency of at least 750 Hz, at a frequency of at least 1000 Hz, at a frequency of 2000 Hz or less, at a frequency of 1500 Hz or less, at a frequency of 1250 Hz or less, at a frequency of 1000 Hz or less, at a frequency of 900 Hz or less, or at a frequency of 800 Hz or less. The method according to claim 12, characterized in that.

16. The step of reducing the pressure of the gas and the step of vibrating the pressure of the gas are performed via an actuating system having an actuating leg coupled to a contact portion of an outer surface of the bladder region aligned with at least a portion of the first inner wall, and the portion of the first inner wall is spaced distally from the sample liquid-gas interface. The method according to claim 6, characterized in that.

17. The portion of the first inner wall is spaced distally from the sample liquid-gas interface by at least 0.2 cm, at least 0.3 cm, at least 0.5 cm, at least 0.75 cm, at least 1.00 cm, at least 1.25 cm, or at least 1.5 cm. The method according to claim 16, characterized in that.

18. The total contact area between the actuating foot and the contact portion is 12 mm 2 or less, 10 mm 2 or less, 8 mm 2 or less, 6 mm 2 or less, 5 mm 2 or less, at least 1 mm 2 at least 2 mm 2 at least 3 mm 2 at least 4 mm 2 or at least 5 mm 2 and is The method according to claim 16, characterized in that.

19. The method is a step of compressing the internal spacing before introducing the sample liquid into the microfluidic channel; a step of maintaining the compression of the internal spacing while introducing the sample liquid into the microfluidic channel; The method according to claim 6, further comprising the above.

20. The step of compressing the internal spacing includes a step of reducing the internal height of the bladder region by at least 40%, at least 50%, at least 60%, at least 65%, at least 75%, at least 80%, at least 85% or at least 90% of the total internal height of the bladder region as measured before the compression, and the internal height is defined by the distance between the first inner wall and the second inner wall when measured along an axis perpendicular to the plane defined by the microfluidic device. The method according to claim 19, characterized in that. ​

21. The step of compressing the internal space includes the step of reducing the internal height of the bladder region by at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 75 μm, at least 85 μm or at least 90 μm, wherein the internal height is defined by the distance between the first inner wall and the second inner wall when measured along an axis perpendicular to the plane defined by the microfluidic device. The method according to claim 19, characterized in that.

22. The total internal height of the bladder region, which is defined by the distance between the first inner wall and the second inner wall when measured along an axis perpendicular to the plane defined by the microfluidic device, is 50 - 200 μm, 75 - 150 μm, 90 - 130 μm, or 110 μm, before the compressing step. The method according to claim 19, characterized in that.

23. The sample liquid and the sample liquid - gas interface are moved to the first zone at a speed of at least 10 μm / s, at least 20 μm / s, at least 50 μm / s, at least 400 μm / s, at least 600 μm / s, at least 750 μm / s, at least 1000 μm / s, at least 1250 μm / s, at least 1500 μm / s, 2000 μm / s or less, 1900 μm / s or less, 1800 μm / s or less, 1500 μm / s or less, 1250 μm / s or less, 1000 μm / s or less, 750 μm / s or less, 500 μm / s or less, 250 μm / s or less, 150 μm / s or less, 100 μm / s or less, or 75 μm / s or less. The method according to claim 1, characterized in that.

24. The volume of the gas being vibrated is in the range of 5 μL to 10 μL, 6.5 μL to 9.0 μL, or 6.9 μL to 8.6 μL. The method according to claim 1, characterized in that.

25. The area of the sample liquid-gas interface is at least 0.03 mm 2 at least 0.04 mm 2 at least 0.06 mm 2 at least 0.07 mm 2 at least 0.08 mm 2 0.25 mm 2 or less, 0.2 mm 2 or less, 0.175 mm 2 or less, 0.15 mm 2 or less, 0.135 mm 2 or less, 0.12 mm 2 or less, or 0.1 mm 2 or less The method according to claim 1, characterized in that.

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