Devices, cartridges and sensors for controlling fluid movement and analyzing biological samples

Abstract

Boxes and devices for operating the boxes to analyze biological samples, such as blood or saliva samples, are described herein. Impedance sensors for analyzing biological samples are also described herein. Methods of determining cell counts or detecting analytes in biological samples are further described herein, which may include conveying the biological sample through a sensor comprising a channel or pore; applying a current or voltage to the channel or pore; detecting impedance within the channel or pore; determining cell counts or detecting analytes based on the detected impedance. Electrowetting electrode arrays are also described herein, which are configured to convey aqueous solutions using low voltages, such as about 50 volts or less. Methods of conveying aqueous liquids using electrowetting electrodes are further described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 680,989, filed on June 5, 2018; U.S. Provisional Patent Application No. 62 / 680,992, filed on June 5, 2018; U.S. Provisional Patent Application No. 62 / 680,993, filed on June 5, 2018; U.S. Provisional Patent Application No. 62 / 756,007, filed on November 5, 2018; and U.S. Provisional Patent Application No. 62 / 756,011, filed on November 5, 2018; the entire disclosure of each of these applications is hereby incorporated by reference into this document. Technical Field

[0003] Some aspects of the invention relate to boxes, systems and methods for analyzing biological samples. Some aspects of the invention relate to sensors for analyzing cells or analytes in biological samples, and boxes and systems including such sensors. The invention also relates to methods of using sensors, boxes and systems, and methods of analyzing biological samples. Systems for controlling the movement of liquids by electrowetting using low voltage electrowetting electrodes, and methods for controlling the movement of liquids by using low voltage electrowetting electrodes are also provided. Background Art

[0004] Blood analysis for accurate medical diagnosis is typically performed in clinical laboratories by specially trained technicians using complex equipment. Blood samples need to be carefully processed by mixing with reagents before analysis by sensors or cell sorters to determine blood cell counts or quantify protein, electrolyte or enzyme concentrations. This specialized training and equipment results in significant patient expense and long delays to obtain important laboratory results. At-home or point-of-care blood analysis systems, such as glucometers, provide fast and convenient diagnostic results but are often limited in accuracy or the range of tests that can be performed.

[0005] Digital microfluidics using electrowetting electrodes is a developing field that explores the actuation of droplets by modulating the hydrophobicity by applying an electric field to the liquid. However, digital microfluidics applications are limited due to the relatively high voltages applied to the electrowetting electrodes to manipulate the liquid.

[0006] There remains a need for devices and systems that provide rapid, accurate and convenient blood analysis systems that do not require special training.

[0007] The disclosures of all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety. If any reference incorporated by reference conflicts with the present disclosure, the present disclosure controls. SUMMARY OF THE INVENTION

[0009] A box for analyzing a biological sample is described herein, comprising: a sample receiving port configured to receive the biological sample; and a sensor configured to analyze the biological sample; a box space fluidly connected to the biological sample receiving port and the sensor; a plurality of electrowetting electrodes configured to transport the biological sample in the box space and combine the biological sample with one or more reagents; and a device interface configured to receive power from and communicate with the box interface on the device, wherein the sensor and the plurality of electrowetting electrodes are electrically connected to the device interface.

[0010] In some embodiments of the box, the box space is fluidly connected to one or more reagent containers. In some embodiments, one or more reagent containers include reagents. In some embodiments of the box, the box space includes a reagent mixing area.

[0011] In some embodiments of the cartridge, the cartridge is a disposable cartridge.

[0012] In some embodiments of the cartridge, the cartridge includes a waste container configured to receive the biological sample after the biological sample is analyzed by the sensor.

[0013] In some embodiments of the cartridge, the biological sample enters the sample receiving port by capillary action.

[0014] In some embodiments of the box, a plurality of electrowetting electrodes include a plurality of coplanar electrodes. In some embodiments, a plurality of electrowetting electrodes also include a ground electrode on a surface opposite to the coplanar electrodes. In some embodiments, the ground electrode is common to two or more electrowetting electrodes. In some embodiments, at least one of the electrowetting electrodes is paired with a separate ground electrode.

[0015] In some embodiments of the box, the electrowetting electrode is coated with an insulating layer. In some embodiments, the insulating layer has a dielectric constant of about 3.9 or higher. In some embodiments, the insulating layer includes hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide or silicon nitride. In some embodiments, the insulating layer is coated on the electrode using atomic layer deposition, chemical vapor deposition, reactive ion beam deposition, sputtering deposition, evaporation, spraying, spin coating or sol-gel formation. In some embodiments, the insulating layer has a thickness of about 1nm to about 5μm. In some embodiments, the insulating layer is a fluid contact surface. In some embodiments, the insulating layer includes a nanostructured surface.

[0016] In some embodiments of the box, the electrowetting electrode is coated with a hydrophobic layer. In some embodiments, the hydrophobic layer is a fluid contact layer. In some embodiments, the hydrophobic layer includes a nanostructured surface. In some embodiments, the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecane hydroxamic acid, stearic acid, octadecanephosphonic acid, 16-hydroxyhexadecane hydroaromatic acid or octadecanethiol. In some embodiments, the hydrophobic layer is coated on the insulating layer.

[0017] In some embodiments of the cartridge, the electrowetting electrode comprises gold, silver, silver chloride, platinum, indium tin oxide, or conductive carbon.

[0018] In some embodiments of the cartridge, the electrowetting electrode is separated from the fluid contacting surface by about 1 nm to about 25 μm.

[0019] In some embodiments of the cartridge, the plurality of electrowetting electrodes are configured to transport the biological sample using a voltage of less than about 50 volts.

[0020] In some embodiments of the cartridge, the plurality of electrowetting electrodes are configured to transport the biological sample using a voltage of about 0.5V to about 50V.

[0021] In some embodiments of the box, the sensor is an impedance sensor. In some embodiments, the sensor is configured to detect protein or measure the amount of protein. In some embodiments, the sensor includes a first sensing electrode functionalized with an affinity portion, and a second sensing electrode paired with the first sensing electrode, wherein the sensor is configured to detect changes in impedance when an analyte or protein is bound to the affinity portion. In some embodiments, the affinity portion is an antibody, an antibody fragment or an aptamer. In some embodiments, the first sensing electrode is an electrowetting electrode. In some embodiments, the sensor also includes a first electrowetting electrode and a second electrowetting electrode, wherein the first electrowetting electrode and the second electrowetting electrode are on opposite sides of the first sensing electrode or the second sensing electrode. In some embodiments, the first sensing electrode is an electrowetting electrode. In some embodiments, a plurality of electrowetting electrodes are configured to statically position at least a portion of a biological sample between the first sensing electrode and the second sensing electrode. In some embodiments, the first electrowetting electrode, the second electrowetting electrode, and the first sensing electrode are electrically connected to a voltage switching circuit, which is configured to selectively activate one of the first electrowetting electrode, the second electrowetting electrode, or the first sensing electrode or not activate any of the first electrowetting electrode, the second electrowetting electrode, or the first sensing electrode. In some embodiments, the voltage switching circuit is electrically connected to a switch, which is configured to selectively select an impedance sensing circuit or an electrowetting electrode power supply circuit. In some embodiments, the impedance sensor includes a pH-sensitive or ion-sensitive layer configured to modulate impedance based on pH or ion concentration. In some embodiments, the impedance sensor includes a metal oxide semiconductor capacitor (MOSCap) sensor. In some embodiments, the MOSCap sensor is adjacent to the first sensing electrode and the second sensing electrode.

[0022] In some embodiments of the cartridge, the cartridge further comprises one or more optical windows.

[0023] In some embodiments of the box, the sensor is a channel sensor, which includes: a first channel segment, a second channel segment, a hole or channel fluidically connecting the first channel segment and the second channel segment, and an electrode pair configured to apply an electric current to the hole or channel and detect the impedance in the hole or channel. In some embodiments, the electrode pair is configured to directly contact the liquid flowing through the channel sensor. In some embodiments, the channel sensor is a flow cytometer configured to count the number of cells in a biological sample. In some embodiments, the sensor is configured to distinguish between different types of cells. In some embodiments, the sensor is configured to distinguish between red blood cells, white blood cells and platelets. In some embodiments, the sensor is configured to count the number of red blood cells, the number of white blood cells or the number of platelets. In some embodiments, the sensor is configured to distinguish eosinophils, basophils, neutrophils, monocytes and lymphocytes. In some embodiments, the sensor is configured to count the number of eosinophils, the number of basophils, the number of neutrophils, the number of monocytes or the number of lymphocytes. In some embodiments, the hole or channel is a micropore or microchannel. In some embodiments, the electric current is a multi-channel current including a plurality of alternating current components at different frequencies. In some embodiments, the electric current is a multi-channel current including (1) a DC component or a low-frequency alternating current, and (2) a plurality of alternating current components at different frequencies. In some embodiments, a plurality of alternating current components include at least three alternating current components. In some embodiments, a plurality of alternating current components include a first alternating current of about 10kHz to about 100kHz, a second alternating current of about 100kHz to about 700kHz, a third alternating current of about 700kHz to about 5MHz, and a fourth alternating current greater than about 5MHz. In some embodiments, a plurality of alternating currents include at least five different alternating current components. In some embodiments, a plurality of alternating current components include a first alternating current of about 50kHz to about 250kHz, a second alternating current of about 250kHz to about 700kHz, a third alternating current of about 700kHz to about 5MHz, a fourth alternating current of about 5MHz to about 20MHz, and a fifth alternating current of about 20MHz to about 150MHz. In some embodiments, the alternating current also includes an additional alternating current of about 100kHz or less or about 50kHz or less. In some embodiments, the electrode pair is configured to detect impedance at a sampling rate of about 100kHz or higher. In some embodiments, the electrode pair is configured to detect the actual impedance component and the imaginary impedance component in the hole or channel. In some embodiments, the electrode pair is configured to detect the amplitude impedance component and the phase impedance component in the hole or channel. In some embodiments, the channel sensor is configured to detect the analyte concentration. In some embodiments, the analyte is a protein.In some embodiments, the box includes a reagent comprising an affinity moiety, and wherein the box is configured to mix the reagent comprising the affinity moiety with the biological sample or a subsample derived from the biological sample, and deliver the biological sample or subsample to the channel sensor. In some embodiments, the hole or channel is a nanopore or nanochannel.

[0024] In some embodiments of the cartridge, the cartridge includes a plurality of sensors.

[0025] In some embodiments of the kit, the biological sample is a blood sample.

[0026] Also provided herein is a system for analyzing a biological sample, comprising: any one of the above-mentioned boxes; and a device including a box interface, the box interface being configured to dock with the box, the device being configured to power the box and operate the box. In some embodiments, the device is configured to power two or more different types of boxes and operate two or more different types of boxes. In some embodiments, the device further includes one or more processors and a non-transitory computer-readable storage medium storing one or more programs configured to be executed by one or more processors, the one or more programs including instructions for the following operations: (a) operating multiple electrowetting electrodes in the box to transport biological samples in the box, and (b) operating sensors. In some embodiments, one or more programs include instructions for operating multiple electrowetting electrodes in the box to continuously transport biological samples through the sensor. In some embodiments, one or more programs include instructions for operating multiple electrowetting electrodes in the box to statically position biological samples in the sensor. In some embodiments, one or more programs include instructions for determining cell counts or analyte concentrations based on multivariate patterns of impedance signals detected at one or more frequencies. In some embodiments, the multivariate pattern includes one or more of impedance peak height, impedance peak width, impedance peak area, or impedance peak half-width peak height. In some embodiments, the multivariate pattern includes a real impedance component and an imaginary impedance component. In some embodiments, the multivariate pattern includes the amplitude of the impedance component and the phase of the impedance component. In some embodiments, one or more programs include instructions for calibrating the sensor. In some embodiments, the device is a handheld device.

[0027] Also provided herein is a method for analyzing a biological sample, comprising: depositing the biological sample into a box; transporting the biological sample in the box using a plurality of electrowetting electrodes; analyzing the biological sample using one or more sensors in the box to generate analytical data; and transmitting the analytical data from the box. In some embodiments, the method includes mixing the biological sample with one or more reagents in the box. In some embodiments, the biological sample is received by the box by capillary action. In some embodiments, the method further includes transporting the biological sample to a waste container in the box after analyzing the biological sample. In some embodiments, the box is disposed of after a single use. In some embodiments, analyzing the biological sample includes counting the number of cells in the biological sample, wherein the analytical data relates to the number of cells. In some embodiments, analyzing the biological sample includes distinguishing between two or more different cell types. In some embodiments, analyzing the biological sample includes distinguishing between red blood cells, white blood cells, and platelets. In some embodiments, analyzing the biological sample includes counting white blood cells, counting red blood cells, or counting platelets. In some embodiments, analyzing the biological sample includes distinguishing between eosinophils, basophils, neutrophils, monocytes, and lymphocytes. In some embodiments, analyzing the biological sample includes counting the number of eosinophils, the number of basophils, the number of neutrophils, the number of monocytes, or the number of lymphocytes. In some embodiments, analyzing the biological sample includes applying an electric current to the biological sample and recording the multiplexed impedance of the electric current. In some embodiments, the electric current is a mixed current including multiple AC components of different frequencies. In some embodiments, the electric current is a mixed current including a DC component and multiple AC components at different frequencies. In some embodiments, the electric current is a mixed current including at least five AC components of different frequencies. In some embodiments, the method includes at least one of the self-calibrating sensors to detect different cell sizes, material properties, and / or concentrations. In some embodiments, analyzing the biological sample includes causing the biological sample to flow continuously through at least one of the sensors during analysis.

[0028] In some embodiments of the method, the biological sample is transported within the cartridge using a voltage of about 50 V or less. In some embodiments, the biological sample is transported within the cartridge using a voltage of about 0.5 V to about 50 V.

[0029] In some embodiments of the method, analyzing the biological sample includes determining the concentration of an analyte or protein within the biological sample. In some embodiments, analyzing the biological sample includes statically placing the biological sample on at least one of the sensors. In some embodiments, the biological sample is statically placed on the sensor using an electrowetting electrode.

[0030] In some embodiments of the method, determining the concentration of an analyte or protein in a biological sample comprises: binding the analyte or protein to an affinity moiety, the affinity moiety binding to an electrode within one of the sensors, and measuring the impedance change resulting from the binding of the analyte or protein to the affinity moiety. In some embodiments, the affinity moiety is an antibody, an antibody fragment, or an aptamer.

[0031] A sensor is also described herein, comprising: a first channel segment; a second channel segment; a hole or channel that fluidly connects the first channel segment and the second channel segment; an electrode pair configured to apply a multi-channel current or voltage to the hole or channel; an electrode pair configured to detect impedances of multiple frequencies within the hole or channel, wherein the detected impedance includes at least a first multivariate impedance mode of a first frequency and a second multivariate impedance mode of a second frequency; wherein the electrode pair configured to apply a multi-channel current and the electrode pair configured to detect impedance are the same electrode pair or different electrode pairs. In some embodiments, the first multivariate impedance mode and the second multivariate impedance mode each include a real impedance component and an imaginary component. In some embodiments, the first multivariate impedance mode and the second multivariate impedance mode each include an amplitude and a phase component of the impedance component.

[0032] A box is also provided herein, comprising the above-mentioned sensor, wherein the box is configured to analyze a biological sample. In some embodiments, the box includes a plurality of electrowetting electrodes configured to transport one or more liquids in the box. In some embodiments, the box is configured to mix a reagent with a biological sample or a subsample derived from the biological sample, and transport the biological sample or the subsample to the sensor. In some embodiments, the box includes a device interface electrically connected to the sensor.

[0033] Also provided herein is a device configured to dock with and operate the above-mentioned box. In some embodiments, the device includes one or more processors and a non-transitory computer-readable storage medium storing one or more programs, the program being configured to be executed by the one or more processors, the one or more programs including for determining cell count or analyte concentration based on detected impedance.

[0034] A system is also described herein, comprising: (a) a box configured to analyze a biological sample, the box comprising: (i) a sensor comprising: a first channel segment; a second channel segment; a hole or channel fluidly connecting the first channel segment and the second channel segment; an electrode pair configured to apply a multi-channel current or voltage to the hole or channel; an electrode pair configured to detect impedance at multiple frequencies in the hole or channel, wherein the electrode pair configured to apply the multi-channel current and the electrode pair configured to detect impedance are the same electrode pair or different electrode pairs; and (ii) a device interface electrically connected to the sensor; and (b) a device configured to dock with the box and operate the box, the device comprising one or more processors and a non-transitory computer-readable storage medium storing one or more programs, the program being configured to be executed by the one or more processors, one or more of which include instructions for determining cell counts or analyte concentrations based on impedance detected at multiple frequencies. In some embodiments, the detected impedance includes a real component and an imaginary component. In some embodiments, the detected impedance includes an amplitude component and a phase component.

[0035] A system is also described herein, comprising: (a) a box configured to analyze a biological sample, the box comprising: (i) a sensor comprising: a first channel segment; and a second channel segment; a hole or channel fluidly connecting the first channel segment and the second channel segment; an electrode pair configured to apply a multi-channel current or voltage to the hole or channel; an electrode pair configured to detect impedance at multiple frequencies in the hole or channel, wherein the electrode pair configured to apply the multi-channel current and the electrode pair configured to detect impedance are the same electrode pair or different electrode pairs; and (ii) a device interface electrically connected to the sensor; (b) a device configured to dock with the box and operate it, the device comprising one or more processors and a non-transitory computer-readable storage medium storing one or more programs, the program is configured to be executed by the one or more processors, one or more of these programs including instructions for determining cell count or analyte concentration based on the detected impedance, wherein the detected impedance includes at least a first multivariate impedance mode at a first frequency and a second multivariate impedance mode at a second frequency. In some embodiments, the first multivariate impedance mode and the second multivariate impedance mode each include a real impedance component and an imaginary component. In some embodiments, the first multivariate impedance mode and the second multivariate impedance mode each include a magnitude impedance component and a phase component.

[0036] In some embodiments, the box includes a plurality of electrowetting electrodes configured to transport one or more liquids within the box. In some embodiments, the box is configured to mix a reagent with a biological sample or a subsample derived from the biological sample and transport the biological sample or subsample to the sensor. In some embodiments, the box includes a device interface electrically connected to the sensor.

[0037] In some embodiments, the electrode pair configured to apply multiple currents and the electrode pair configured to detect impedance are the same electrode pair. In some embodiments, the electrode pair configured to apply multiple currents and the electrode pair configured to detect impedance are different electrode pairs.

[0038] In some embodiments, the multivariate impedance pattern includes one or more of impedance peak height, impedance peak width, impedance peak area, or impedance peak half-width peak height.

[0039] In some embodiments, the electrode pair is in direct contact with the interior of the channel.

[0040] In some embodiments, the sensor is configured to count the number of cells in the biological sample. In some embodiments, the sensor is configured to distinguish between different types of cells. In some embodiments, the sensor is configured to distinguish between red blood cells, white blood cells and platelets. In some embodiments, the sensor is configured to count the number of red blood cells, the number of white blood cells or the number of platelets. In some embodiments, the sensor is configured to distinguish between eosinophils, basophils, neutrophils, monocytes and lymphocytes. In some embodiments, the sensor is configured to count the number of eosinophils, basophils, neutrophils, monocytes and lymphocytes.

[0041] In some embodiments, the pores or channels are micropores or microchannels.

[0042] In some embodiments, the sensor is configured to detect an analyte concentration. In some embodiments, the analyte is a protein. In some embodiments, the pore or channel is a nanopore or nanochannel.

[0043] In some embodiments, an electrode pair configured to detect impedance includes a first electrode within a first channel segment and a second electrode within a second channel segment.

[0044] In some embodiments, the sensor includes a channel, wherein an electrode pair configured to detect impedance is located within the channel. In some embodiments, the electrode pair includes a first electrode adjacent to a first channel segment and a second electrode adjacent to a second channel segment. In some embodiments, the electrode pair includes a first electrode on an upper surface of the channel and a second electrode on a lower surface of the channel, and wherein the first electrode is located above the second electrode.

[0045] In some embodiments, the sensor further comprises an electrode pair configured to detect liquid flow within the sensor.

[0046] In some embodiments, the sensor includes one or more isolated electrodes.

[0047] In some embodiments, the sensor comprises an inlet of a first channel segment near the electrowetting electrode. In some embodiments, the sensor comprises an outlet of a second channel segment near the electrowetting electrode.

[0048] In some embodiments, the multi-channel current includes multiple AC components at different frequencies. In some embodiments, the multi-channel current includes (1) a DC component or a low-frequency AC, and (2) multiple AC components at different frequencies. In some embodiments, multiple AC components include at least three AC components. In some embodiments, multiple AC components include a first AC of about 10kHz to about 100kHz, a second AC of about 100kHz to about 700kHz, a third AC of about 700kHz to about 5MHz, and a fourth AC greater than about 5MHz. In some embodiments, multiple AC components include at least five AC components. In some embodiments, multiple AC components include a first AC of about 50kHz to about 250kHz, a second AC of about 250kHz to about 700kHz, a third AC of about 700kHz to about 5MHz, a fourth AC of about 5MHz to about 20MHz, and a fifth AC of about 20MHz to about 150MHz. In some embodiments, the current also includes an additional AC of about 100kHz or less, for example, about 50kHz or less.

[0049] In some embodiments, the electrode pairs configured to detect impedance are configured to detect impedance at a sampling rate of about 100 kHz or higher.

[0050] Also provided herein is a method for determining cell count in a biological sample, the method comprising: conveying the biological sample through a sensor comprising a channel or a hole; applying a multi-channel current or voltage to the channel or the hole; detecting a multiplexed impedance within the channel or the hole; and determining the cell count based on the detected multiplexed impedance. In some embodiments, the detected multiplexed impedance includes a real component and an imaginary component at multiple different frequencies. In some embodiments, the detected multiplexed impedance includes an amplitude component and a phase component at multiple different frequencies.

[0051] Also provided herein is a method for determining cell count in a biological sample, the method comprising: transporting a biological sample through a sensor comprising a channel or a hole; applying a multi-channel current or voltage to the channel or the hole; detecting a multiplexed impedance in the channel or the hole, the multiplexed impedance comprising at least a first multi-element impedance mode at a first frequency and a second multi-element impedance mode at a second frequency; determining the cell count according to the detected multiplexed impedance. In some embodiments, the first multi-element impedance mode and the second multi-element impedance mode each include a real impedance component and an imaginary component. In some embodiments, the first multi-element impedance mode and the second multi-element impedance mode each include an amplitude impedance component and a phase component. In some embodiments, the multi-element impedance mode includes one or more of an impedance peak height, an impedance peak width, an impedance peak area, or an impedance peak half-width peak height. In some embodiments, the biological sample directly contacts a pair of electrodes for detecting multiplexed impedance. In some embodiments, the method includes distinguishing two or more different types of cells. In some embodiments, determining the cell count includes determining a red blood cell count, a white blood cell count, or a platelet count in the biological sample. In some embodiments, determining the cell count includes determining an eosinophil count, a basophil count, a neutrophil count, a monocyte count, and a lymphocyte count in the biological sample. In some embodiments, the method includes passing two or more subsamples of the biological sample through the sensor, wherein the two or more subsamples are processed at different pH levels or different electrolyte concentrations.

[0052] In some embodiments of the above methods, the method includes delivering a marker to the sensor. In some embodiments, the marker is an air bubble or a low conductivity solution. In some embodiments, the method includes detecting the marker. In some embodiments, the marker triggers the start or end of recording of the detected multiplexed impedance. In some embodiments, the method includes determining the flow rate of the biological sample. In some embodiments, the method includes using the determined flow rate to determine the cell count.

[0053] In some embodiments of the above methods, the method comprises filtering the biological sample.

[0054] In some embodiments of the above method, the multi-channel current includes multiple AC components at different frequencies. In some embodiments, the multi-channel current includes (1) a DC component or a low-frequency AC, and (2) multiple AC components at different frequencies. In some embodiments, the multiple AC components include at least three AC components. In some embodiments, the multiple AC components include a first AC of about 10kHz to about 100kHz, a second AC of about 100kHz to about 700kHz, a third AC of about 700kHz to about 5MHz, and a fourth AC greater than about 5MHz. In some embodiments, the multiple AC components include at least five AC components. In some embodiments, the multiple AC components include a first AC of about 50kHz to about 250kHz, a second AC of about 250kHz to about 700kHz, a third AC of about 700kHz to about 5MHz, a fourth AC of about 5MHz to about 20MHz, and a fifth AC of about 20MHz to about 150MHz. In some embodiments, the current also includes an additional AC component of about 100kHz or lower, for example, about 50kHz or lower.

[0055] Also provided herein is a method for detecting an analyte in a biological sample, the method comprising: transporting the biological sample through a sensor comprising a channel or a hole, the biological sample comprising an analyte bound to an affinity portion; applying an electric current or voltage to the channel or hole; detecting the impedance within the channel or hole; and detecting the analyte according to the detected impedance. In some embodiments, the analyte is a protein or an electrolyte. In some embodiments, the affinity portion is a multivalent affinity portion. In some embodiments, the affinity portion is charged. In some embodiments, the affinity portion comprises an aptamer, an antibody, or an antibody fragment. In some embodiments, the affinity portion comprises an aptamer bound to avidin or an avidin derivative. In some embodiments, the method comprises mixing the biological sample with a reagent comprising an affinity portion. In some embodiments, detecting the analyte based on the detected impedance comprises distinguishing between an affinity portion bound to the analyte and an unbound affinity portion.

[0056] In some embodiments of the above method, the method includes delivering a marker to the sensor. In some embodiments, the marker is a bubble or a low conductivity solution. In some embodiments, the method includes detecting the marker. In some embodiments, the marker triggers the start or end of recording of the detected multiplexed impedance. In some embodiments, the method includes determining the flow rate of the biological sample. In some embodiments, the method includes using the determined flow rate to determine the concentration of the analyte.

[0057] Also described herein is an electrowetting electrode array, comprising: a plurality of coplanar electrowetting electrodes, which are coated with an insulating layer and separated by an insulating layer; and wherein the electrowetting array comprises a hydrophobic liquid contact surface, and wherein the electrowetting array is configured to transport an aqueous liquid using a voltage of about 50 volts or less. In some embodiments, the insulating layer comprises a hydrophobic liquid contact surface. In some embodiments, the insulating layer comprises a nanostructured surface. In some embodiments, the insulating layer is coated with a hydrophobic layer comprising a hydrophobic liquid contact surface. In some embodiments, the hydrophobic layer comprises a nanostructured surface. In some embodiments, the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecane hydroxamic acid, stearic acid, octadecanephosphonic acid, 16-hydroxyhexadecane hydroaromatic acid or octadecanethiol. In some embodiments, a plurality of electrowetting electrodes are configured to transport an aqueous liquid using a voltage of about 0.5 volts to about 50 volts.

[0058] In some embodiments, the insulating layer has a dielectric constant of about 3.9 or higher. In some embodiments, the insulating layer comprises hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide or silicon nitride. In some embodiments, the insulating layer is coated on the electrode using atomic layer deposition, chemical vapor deposition, reactive ion beam deposition, sputtering deposition, evaporation, spraying, spin coating or sol-gel formation. In some embodiments, the insulating layer has a thickness of about 1 nm to about 5 μm.

[0059] In some embodiments, the electrowetting electrode comprises gold, silver, silver chloride, platinum, indium tin oxide, or conductive carbon.

[0060] In some embodiments, the electrowetting electrode is separated from the hydrophobic liquid contacting surface by about 1 nm to about 25 μm.

[0061] In some embodiments, the plurality of electrowetting electrodes further comprises a ground electrode parallel to the coplanar electrowetting electrodes, wherein the hydrophobic liquid contact surface is between the ground electrode and the coplanar electrowetting electrodes. In some embodiments, the ground electrode is common to two or more electrowetting electrodes. In some embodiments, at least one of the electrowetting electrodes is paired with a separate ground electrode.

[0062] In some embodiments, the electrowetting electrode array includes an impedance sensor, which includes a sensing electrode and a functionalized liquid contact surface, wherein the functionalized liquid contact surface is functionalized with an affinity portion that specifically binds to a target analyte. In some embodiments, the sensor also includes a second sensing electrode paired with the first sensing electrode, wherein the sensor is configured to detect changes in impedance after the analyte or protein binds to the affinity portion. In some embodiments, the target analyte is a protein. In some embodiments, the affinity portion is an antibody, an antibody fragment, or an aptamer. In some embodiments, the impedance sensor also includes a metal oxide semiconductor capacitor (MOSCap) sensor, which includes a pH-sensitive layer or an ion-sensitive layer configured to modulate impedance based on pH or ion concentration.

[0063] In some embodiments, the aqueous liquid comprises a biological sample. In some embodiments, the biological sample comprises a blood sample.

[0064] Also provided herein is a box for analyzing a biological sample, comprising: a sample receiving port configured to receive the biological sample; and a sensor configured to analyze the biological sample; a chamber or channel in fluid communication with the biological sample receiving port and the sensor, the chamber or channel comprising the above-mentioned electrowetting electrode array; and a device interface configured to receive power from the box interface on the device and communicate with it, wherein the sensor and a plurality of electrowetting electrodes are electrically connected to the device interface. In some embodiments, the chamber or channel is fluidically connected to one or more reagent containers, wherein the electrowetting electrode array extends into one or more reagent containers. In some embodiments, the electrowetting electrode array includes a reagent mixing area.

[0065] The present invention also provides a system for analyzing a biological sample, the system comprising: the above-mentioned cartridge; and a device comprising a cartridge interface, the cartridge interface being configured to dock with the cartridge, the device being configured to operate the electrowetting electrode array. In some embodiments, the device is a handheld device.

[0066] Also provided herein is a method for transporting a liquid, the method comprising: placing an aqueous liquid on a first hydrophobic liquid contact surface above an inactivated first electrowetting electrode; activating the second electrowetting electrode by applying a voltage of about 50 volts or less to the second electrowetting electrode, thereby transporting the aqueous liquid from the first hydrophobic liquid contact surface to a second hydrophobic liquid contact surface above the second electrowetting electrode; wherein the first electrowetting electrode and the second electrowetting electrode are covered and separated by an insulating layer. In some embodiments, the aqueous liquid comprises a biological sample.

[0067] In some embodiments, the method includes delivering a reagent to the second liquid contacting surface, thereby mixing the reagent with the aqueous liquid. In some embodiments, the reagent is delivered by activating the second electrowetting electrode.

[0068] In some embodiments of the above methods, a voltage of about 0.5 V to about 50 V is used to transport the biological sample within the cartridge.

[0069] In some embodiments of the above methods, the first hydrophobic liquid contacting surface or the second hydrophobic liquid contacting surface is a nanostructured surface.

[0070] In some embodiments of the foregoing methods, the insulating layer includes a first hydrophobic liquid contacting surface and a second hydrophobic liquid contacting surface.

[0071] In some embodiments of the above method, the insulating layer is coated with a hydrophobic layer comprising a first hydrophobic liquid contact surface and a second hydrophobic liquid contact surface. In some embodiments, the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecanehydroxamic acid, stearic acid, octadecanephosphonic acid, 16-hydroxyhexadecanehydroaromatic acid, or octadecanethiol.

[0072] In some embodiments of the above methods, the insulating layer has a dielectric constant of about 3.9 or higher. In some embodiments, the insulating layer includes hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide, or silicon nitride.

[0073] In some embodiments of the above method, the insulating layer has a thickness of about 1 nm to about 5 μm.

[0074] In some embodiments of the above methods, the first electrowetting electrode is separated from the first hydrophobic liquid contacting surface by about 1 nm to about 25 μm, and the second electrowetting electrode is separated from the second hydrophobic liquid contacting surface by about 1 nm to about 25 μm. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1A An exemplary cartridge for analyzing a biological sample is shown.

[0076] Figure 1B Another exemplary embodiment of a cartridge is shown.

[0077] Figure 2 A block diagram of an exemplary device that interfaces with a cartridge is shown.

[0078] Figure 3 An exemplary impedance sensor pair configured to measure analyte concentration is shown.

[0079] Figure 4A An exemplary impedance sensor integrated with a plurality of electrowetting electrodes configured to detect or measure an analyte is shown.

[0080] Figure 4B Shows Figure 4A In an impedance sensor, a hydrophobic layer covers the insulating layer of the sensor.

[0081] Figure 5A An exemplary impedance sensor that can be used for indirect detection or quantification of an analyte is shown. The impedance sensor includes a MOSCap sensor.

[0082] Figure 5B Shown as Figure 5A FIG. 4 is a side view of a MOSCap sensor shown in FIG. 4 , and an electrical model for the impedance sensor. FIG. 4 is a side view of a MOSCap sensor shown in FIG. 4 .

[0083] Figure 6 Shows the use of Figure 5A and 5B Direct and indirect analyte measurements of the sensor are shown.

[0084] Figure 7 An image of an exemplary channel of a channel sensor having a first channel segment, a second channel segment, and a microchannel between the first channel segment and the second channel segment is shown.

[0085] Fig. 8A A top view and a side view of a channel sensor having a first impedance detection electrode in a first channel segment and a second impedance detection electrode in a second channel segment with a microwell or microchannel between the first channel segment and the second channel segment are shown.

[0086] Figure 8B A top view and a side view of a channel sensor are shown, the channel sensor having a first impedance detection electrode adjacent to a first channel segment within a microchannel, and a second impedance detection electrode adjacent to a second channel segment within the microchannel.

[0087] Figure 8C A top view and a side view of a channel sensor are shown. The channel sensor includes a first impedance detection electrode and a second impedance detection electrode located in a microchannel, wherein the first impedance detection electrode is located above the second impedance detection electrode.

[0088] Fig.8D An exemplary channel sensor having two sets of detection electrodes within the same channel is shown.

[0089] Fig. 8E Another exemplary channel sensor is shown having two sets of detection electrodes in the same channel and having a wavy channel feature in the channel at each electrode location.

[0090] Figure 8FAn exemplary embodiment of a microchannel between a first channel segment and a second channel segment is shown, wherein a plurality of sensing electrodes are disposed along the length of the channel.

[0091] Fig. 9 A top view and a side view of a channel sensor including a pair of impedance detection electrodes, two pairs of flow detection electrodes, and two isolation electrodes are shown.

[0092] Fig.10 Showing the start of operation of the channel sensor, the lower curve demonstrates the DC voltage detected by the first pair of flow detection electrodes.

[0093] Fig.11 It is shown how a marker can be used as a trigger for data collection using a sensor, and the cycle can be repeated for different biological subsamples.

[0094] Fig.12 An exemplary method of assaying a biological sample using a channel sensor in a cartridge is shown.

[0095] Fig.13 An electrical model of a cell flowing through a pore or channel is shown.

[0096] Fig.14 Exemplary relative impedance changes as a function of time, measured by an impedance sensing electrode at a single frequency (30 kHz), are shown for a cell of approximately 10 μm.

[0097] Fig.15A Binding of target analyte to a tetravalent affinity molecule is shown.

[0098] Fig. 15B Impedance changes caused by affinity moieties not bound to the target analyte (upper graph) and impedance changes caused by affinity moieties bound to the target analyte (lower graph) as the affinity moieties pass through the nanochannel of the channel sensor are shown.

[0099] Fig. 15C One example of using impedance magnitude to determine the concentration of a target analyte is shown.

[0100] Fig.16A An exemplary electrowetting electrode array is shown having an 8x16 matrix with traces electrically connecting each electrode to the device interface.

[0101] Fig. 16B Shows Fig.16A A magnified view of several electrowetting electrodes in the array is shown, highlighting the jagged edges of the approximately square electrodes.

[0102] Fig.17A One embodiment of a plurality of electrowetting electrodes comprising an insulating layer fluid contacting surface is shown.

[0103] Fig. 17B Another embodiment of a plurality of electrowetting electrodes is shown, which includes an insulating layer coating the electrowetting electrodes and a hydrophobic layer coating the insulating layer.

[0104] Fig. 17C Another embodiment of a plurality of electrowetting electrodes comprising an insulating layer fluid contacting surface is shown.

[0105] Fig.17D Another embodiment of a plurality of electrowetting electrodes is shown, which includes an insulating layer coating the electrowetting electrodes and a hydrophobic layer coating the insulating layer.

[0106] Fig.18A The voltage change in a flow cytometer as a 30 kHz current flows through polystyrene beads of 5 μm, 10 μm, 20 μm or 30 μm in diameter. When the current is passed through the beads, a significant voltage drop is observed, which can be used to measure the particle size.

[0107] Fig.18B A normalized curve of the diameter of polystyrene beads measured using flow cytometry (30 kHz current) versus the actual size of the polystyrene beads is shown. The measured diameter is linearly related to the actual diameter, indicating that this method can be reliably used to estimate particle size.

[0108] Fig.19 Shown are the real and imaginary components of the multiplexed impedance measured for an eosinophil as it passes current at -6.19 seconds, at currents having 107 kHz, 570 kHz, 1.55 MHz, 16 MHz, and 40 MHz components.

[0109] Fig. 20A -G shows basophils ( Fig. 20A ), eosinophils ( Fig. 20B ), lymphocytes ( Fig. 20C ), monocytes ( Fig.20D ), neutrophils ( Fig.20E ), platelets ( Fig.20F ) and red blood cells ( Figure 20G ) Average normalized impedance plot of the real and imaginary components at various currents (107kHz, 570kHz, 1.55MHz, 16MHz and 40MHz).

[0110] Fig.21A Shown are the normalized voltage difference signals from 5 μm and 10 μm polystyrene beads flowing through the channel sensor.

[0111] Fig. 21BA histogram of bead counts versus measured voltage difference is shown for 5 μm and 10 μm polystyrene beads flowing through a channel sensor. The results show that polystyrene beads can be sorted by size based on the impedance attributed to the beads passing through the channel sensor.

[0112] Fig.22A Clustering of white blood cells (WBC), red blood cells (RBC), and platelets by a machine learning model that classifies particles based on impedance features is shown.

[0113] Fig.22A Clustering of neutrophils, lymphocytes, and monocytes by a machine learning model that classifies particles based on impedance signatures is shown.

[0114] Fig. 22C The component voltage differential signals are shown as a function of time as diluted whole blood passes through the sensor. Using a machine learning model, particles passing through the sensor are labeled as red blood cells (R), white blood cells (W), platelets (P), or polystyrene beads (PS) based on their attributed impedance signature. DETAILED DESCRIPTION OF THE INVENTION

[0116] Boxes and systems for analyzing biological samples (e.g., blood, serum, saliva, sweat, tears, mucus, urine, or any other biological sample or derivative suspended in a fluid) and methods for analyzing biological systems are described herein. Different types of boxes can be used together with control devices, and different types of boxes can be configured to perform different determinations or determination sets of biological samples. For example, in some embodiments, the box is configured to perform a complete blood cell count (CBC) assay. The CBC assay can distinguish and count different blood cell types, such as red blood cells, platelets, and white blood cells (including eosinophils, basophils, neutrophils, monocytes, and lymphocytes). In some embodiments, the box is configured to perform a comprehensive metabolic set (CMP). The same control device can be engaged with different boxes, thereby allowing the use of a universal device that can perform multiple determinations by docking with a selected box.

[0117] The box includes: a sample receiving port that can receive a biological sample; a sensor for analyzing the sample; and a box space that connects the biological sample receiving port fluid to the sensor. Multiple electrowetting electrodes can transport the biological sample in the box space, and can optionally divide the sample into smaller volumes and / or further combine the biological sample with one or more reagents to process the biological sample. The box can be docked with the device through a device interface on the box, which is configured to receive power from the device and communicate with the device. The sensor and multiple electrowetting electrodes are electrically connected to the device interface, thereby allowing the device to operate the box.

[0118] The sensor is used to analyze the biological sample, and can be, for example, a sensor configured to detect proteins (which can be, for example, enzymes) or electrolytes or to count the number and / or type of cells in the biological sample. In some embodiments, the box includes two, three, four or more sensors that can have the same or different functions. For example, the box can include a first sensor for detecting proteins and a second sensor for counting the number of cells, or a hybrid sensor that can detect proteins and count the number of cells.

[0119] The biological sample can be analyzed by depositing the biological sample into the box and transporting the biological sample in the box using multiple electrowetting electrodes. For example, the biological sample of a fixed volume is directly placed in the input port or the biological sample is introduced into the box through an interface that allows the placement of a blood collection tube, thereby forming a fluid path from the tube to the input port. In some embodiments, the biological sample is prepared by dividing the blood sample into multiple droplets of a predetermined volume. The sample can be mixed with one or more reagents in the box to process the biological sample, and one or more sensors in the box are used to analyze it to generate analytical data. The analytical data is then transmitted to the device.

[0120] Multiple electrowetting electrodes in the cartridge allow the biological sample to be carefully transported within the cartridge using low voltage forces. The cartridge may include a reagent container, and the reagents within the container may be mixed with the biological sample on the electrowetting electrodes before being transported to the sensor for analysis.

[0121] The cartridge is preferably a disposable cartridge that includes the necessary reagents and correct volumes to process the biological sample without user intervention. The user simply applies the biological sample to the cartridge and the device operates the cartridge to properly process and analyze the sample. Once the analysis is complete, the cartridge can be disposed of. Different cartridge types can be manufactured, including different analytical tests, depending on the sensor or sensors included in the cartridge, and a single device can interface with different cartridge types.

[0122] Also described herein is a sensor for analyzing a biological sample using multiplexed impedance sensing. Biological samples can be analyzed, for example, to detect or quantify proteins (which can be, for example, enzymes) or electrolytes, or to count the number and / or type of cells (such as red blood cells, white blood cells, or platelets). Such biological samples can include, for example, blood, serum, saliva, sweat, tears, mucus, urine, or any other biological sample or derivative suspended in a fluid. In some embodiments, the sensor is included in a box that is configured to receive and analyze the biological sample. The box can be a part of a system that receives and operates a box.

[0123] In some embodiments, the sensor includes an electrode pair configured to detect impedance at multiple frequencies within a hole or channel. The detected impedance may include a multivariate impedance pattern at one or more frequencies, and one or more multivariate impedance patterns may be analyzed to count the number of cells and / or distinguish cell types. The multivariate impedance pattern may include, for example, a signal (such as amplitude) from a real component of the impedance and a signal (such as amplitude) from an imaginary component of the impedance. Alternatively, the multivariate impedance pattern may include, for example, a signal (such as amplitude) from an amplitude component of the impedance and a signal (such as amplitude) from a phase component of the impedance.

[0124] Further disclosed herein is a method for determining cell count in a biological sample. Cell counting can include counting a variety of different types of cells, such as counting of white blood cells, counting of red blood cells and / or counting of platelets in a biological sample. In some embodiments, cell counting can include white blood cell differences, and can include counting of basophils, counting of eosinophils, counting of lymphocytes, counting of neutrophils and / or counting of monocytes. The method can include conveying a biological sample (or subsample) through a sensor including a channel or a hole. A multi-channel current or voltage is applied to the channel or hole, and a multiplexed impedance can be detected in the channel or hole. The multiplexed impedance includes at least a first multivariate impedance mode at a first frequency and a second multivariate impedance mode at a second frequency. Cell count can then be determined based on the detected multiplexed impedance. As further explained herein, when cells or other particles flow through a channel or hole, the multiplexed impedance mode can include the characteristics of the detected impedance signal. Such characteristics can include, but are not limited to, one or more of impedance peak height, impedance peak width, impedance peak area or impedance peak half-peak height. The feature can come from the real component or imaginary component of the impedance signal. The characteristic can come from the magnitude component or the phase component of the impedance signal.

[0125] Also described herein is a method for detecting an analyte such as a protein or electrolyte in a biological sample. The method includes conveying the biological sample through a sensor including a channel or hole, applying an electric current or voltage to the channel or hole, and detecting the impedance in the channel or hole. The biological sample includes an analyte bound to an affinity portion, and when the analyte bound to the affinity portion passes through the channel or hole, the impedance is affected. Therefore, the analyte can be detected based on the detected impedance.

[0126] In some optional embodiments of the electrowetting electrode array described herein, the electrowetting electrode array is configured to transport aqueous liquids (e.g., biological samples or processed biological samples) by applying a low voltage (about 50 volts or less) to the electrowetting electrodes. The voltage applied to the electrodes causes a change in the liquid contact angle, thereby driving the liquid. By using a low voltage to transport aqueous liquids, the electrowetting electrode array can be used in low-power devices and applications, such as instant measurement systems. The instant measurement system can include a box with an electrowetting electrode array, which is configured to analyze biological samples. The box includes an electrowetting array in the box, which can be operated by a device, which can be a handheld and / or battery-powered device.

[0127] In some embodiments, the electrowetting electrode array comprises a plurality of coplanar electrowetting electrodes coated with and separated by an insulating layer; wherein the electrowetting array comprises a hydrophobic liquid contact surface, and wherein the electrowetting array is configured to transport an aqueous liquid using a voltage of about 50 volts or less. As further described herein, the hydrophobicity (i.e., contact angle) of the liquid contact surface, and the distance and / or material separating the liquid contact surface and the electrode are selected to reduce the voltage required to transport an aqueous liquid from one electrowetting electrode to another electrowetting electrode sample.

[0128] Liquid can be transported by placing an aqueous liquid on a first hydrophobic liquid contact surface above an inactivated first electrowetting electrode, and activating the second electrowetting electrode by applying a voltage of about 50 volts or less to the second electrowetting electrode, thereby transporting the aqueous liquid from the first hydrophobic liquid contact surface to a second hydrophobic liquid contact surface above the second electrowetting electrode. The first electrowetting electrode and the second electrowetting electrode are coated with an insulating layer and separated by an insulating layer.

[0129] The box for analyzing biological samples (e.g., blood, serum, saliva, sweat, tears, mucus, urine, or any other biological sample or derivative suspended in a fluid) may include an electrowetting electrode array to transport and / or process the biological sample in the box. The box may further include one or more sensors to analyze the biological sample. Different types of boxes can be used with the control device, and different types of boxes can be configured to perform different determinations or determination sets of biological samples. The same control device can be engaged with different boxes, thereby allowing the use of a universal device that can perform multiple determinations by docking with a selected box.

[0130] definition

[0131] As used herein, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0132] References herein to "about" or "approximately" a value or parameter include (and describe) variations to that value or parameter itself. For example, description referring to "about X" includes description of "X."

[0133] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), double antibodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, bispecific antibodies, functionally active epitopes-binding fragments thereof, bifunctional hybrid antibodies, single-chain antibodies, and Fc-containing polypeptides, such as immunoadhesion. In some embodiments, the antibody can be any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD). In some embodiments, the antibody can be any light chain isotype (e.g., κ or γ). The antibody can be non-human (e.g., from a mouse, goat, or any other animal), fully human, humanized, or chimeric. In some embodiments, the antibody is a derived antibody.

[0134] The term "channel" refers to a tube, conduit, or other passageway that provides a linear path for liquid flow.

[0135] It should be understood that aspects and variations of the present invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0136] A "high-κ" or "high-κ" material is any material with a dielectric constant of 3.9 or higher.

[0137] A "hydrophobic" material, surface or layer is any material, surface or layer that provides a water contact angle of 90 degrees or greater at 25°C.

[0138] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, as well as any other specified value or intervening value in the specified range, is included within the scope of the present disclosure. Where the stated range includes an upper or lower limit, ranges excluding those included upper limits are also included in the present disclosure.

[0139] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention.The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0140] The features and preferences described above with respect to the "embodiment" are different preferences and are not limited to this particular embodiment; where technically feasible, they can be freely combined with features from other embodiments, and can form a preferred combination of features. The description is given to enable a person skilled in the art to make and use the invention, and the description is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles herein can be applied to other embodiments. Therefore, the present invention is not intended to be limited to the embodiments shown, but is consistent with the widest scope consistent with the principles and features described herein.

[0141] Analysis box

[0142] The box receives a biological sample that can be placed by a user, and processes and analyzes the biological sample. The box includes: a sample receiving port configured to receive a biological sample; a sensor for analyzing the biological sample; a chamber or channel connected to the biological sample receiving port and the sensor fluid; a plurality of electrowetting electrodes configured to transport the biological sample in the chamber or channel and combine the biological sample with one or more reagents; and a device interface configured to receive power from and communicate with the box interface on the device, wherein the sensor and the plurality of electrowetting electrodes are electrically connected to the device interface. The box is preferably a disposable box, and a new box can be used to analyze different biological samples. In some embodiments, the plurality of electrowetting electrodes are configured to (1) transport the biological sample in the chamber or channel, and (2) combine the biological sample with one or more reagents in the box.

[0143] Electrowetting electrodes can be used to perform fluid actuation within the box, although other forms of liquid (e.g., sample) delivery within the box are contemplated. For example, in some embodiments, the box includes one or mechanical pumps (e.g., syringe pumps, peristaltic pumps, and / or pneumatic pumps) to deliver liquid within the box. One or more mechanical pumps can, for example, be used in conjunction with one or more electronic fluid valves. Therefore, although certain embodiments of the boxes described herein use electrowetting electrodes for liquid movement and / or sample preparation, other liquid delivery methods can be used.

[0144] Exemplary biological samples that can be deposited into the box include, but are not limited to, blood, plasma, serum, sweat, tears, mucus, urine, or any other suitable liquid biological sample or biological sample derivative. In some embodiments, the sample has a volume of about 20 μL or more, such as a volume between about 20 μL and about 1 mL. For example, in some embodiments, the volume of the sample is about 20 μL to about 40 μL, about 40 μL to about 80 μL, about 80 μL to about 150 μL, about 150 μL to about 300 μL, about 300 μL to about 600 μL, or about 600 μL to about 1 mL. The biological sample received by the box can be separated in the box to perform different tests or perform redundant tests. The biological sample can be segmented and / or metered (i.e., reaching a predetermined volume) using an electrowetting electrode. In some embodiments, the sample is separated to perform 2, 3, 4 or more redundant tests (i.e., repeat, three times, etc.). In some embodiments, the sample is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different tests, each test may or may not be repeated. For example, 600 μL of biological sample can be divided into 30 separate samples, each separate biological sample has 20 μL of biological sample, and three replicates can be analyzed using 10 different analytical tests.

[0145] The sample receiving port may include an inlet fluidically connected to the sample receiving chamber by a first sample port channel, and the sample receiving chamber may be fluidically connected to a chamber or channel of the box by a second sample port channel. The volume of the sample receiving chamber determines the volume of the sample received by the box. Therefore, in some embodiments, the volume of the sample receiving chamber is about 20 μL or larger, for example, between about 20 μL and about 1 mL. For example, in some embodiments, the volume of the sample receiving chamber is about 20 μL to about 40 μL, about 40 μL to about 80 μL, about 80 μL to about 150 μL, about 150 μL to about 300 μL, about 300 μL to about 600 μL or about 600 μL to about 1 mL. The sample deposited in the inlet flows into the sample receiving chamber through the first sample port channel by capillary action. The sample is retained in the sample receiving chamber until a plurality of electrowetting electrodes are operated to transport the sample from the sample receiving chamber. As further discussed herein, the electrowetting electrode is preferably coated with a material that provides a hydrophobic fluid contact surface that prevents the sample from flowing into a chamber or channel of the device through the second sample port channel without electrowetting actuation. The second sample port channel and / or the sample receiving chamber may optionally include one or more electrowetting electrodes that can help transfer the biological sample from the sample receiving chamber to the main chamber or channel. Also optionally, the box may include a filter that is configured to filter the biological sample before it enters the main chamber. The filter can, for example, remove debris or larger particles from the biological sample before the sample is processed and analyzed in the box. The filter may be positioned in, for example, the second sample port channel or the first sample port channel.

[0146] The sample receiving port fluid is connected to the main chamber or channel, and the main chamber or channel is connected to one or more sensors or other box parts fluid. If the box includes a main channel for transporting a biological sample in the whole box, the size of the channel is preferably set to limit the capillary motion of the sample in the channel. The bottom of the chamber or channel includes a plurality of electrowetting electrodes, which can be coated as described herein. The main chamber or channel also includes a top and a sidewall to surround the channel or chamber, although the sidewall can be optionally rounded to avoid corners. A plurality of electrowetting electrodes are configured to actuate the biological sample in the chamber or channel, for example, to a reagent mixing area, a sensor or a waste container by electrowetting. In some configurations, a plurality of electrowetting electrodes are located at the bottom of the chamber or channel, and in some embodiments, a plurality of electrowetting electrodes are located at the bottom and top of the chamber or channel. Contrary to the channel forcing the linear motion of the fluid, having a main chamber in a box with an electrowetting array allows the motion of the fluid in the box to be programmatically controlled using electrowetting electrodes.

[0147] The chamber or channel can be fluidly connected with one or more reagent containers, and the reagent containers can be pre-loaded with reagents to process biological samples. The size of the reagent container can be set to accommodate the desired amount of reagents. The reagent container is connected to the main chamber or channel by a container channel. One or more reagent containers and / or container channels can optionally include one or more electrowetting electrodes to transport reagents from the reagent chamber to the main chamber or channel. The electrowetting electrode optionally transports the sample and the reagent to the reagent mixing area, thereby combining the biological sample with one or more reagents to form a processed biological sample. The reagent can be used to, for example, change pH, perform chemical and / or biological reactions, or dilute (or serially dilute) biological samples to form processed biological samples. In some embodiments, one or more reagents are configured to modify or crack cells in the sample or a subset of cells in the sample. For example, in some embodiments, the cells in the lysing agent cracking sample, or only the red blood cells in the cracking sample. Targeted lysis of red blood cells in the sample is optionally performed, for example, to count the white blood cells in the sample or measure hemoglobin content or concentration with the processing sample. In some embodiments, one or more agents can modify the cell surface (e.g., cell membrane) with a marker (e.g., an affinity moiety marker that can be labeled, such as an aptamer or antibody (or fragment)), which specifically binds to the cell type for identification. In some embodiments, one or more agents include a marker that can permeate into a cell or a targeted subset of cells, which can modify the interior of the cell (e.g., the cytoplasm or nucleus), which can be used for cell type identification.

[0148] In some embodiments, one or more reagents include a buffer, an anticoagulant (e.g., ethylenediaminetetraacetic acid (EDTA)) or a stabilizer (e.g., a stabilizer protein, such as albumin) and / or a viscosity modifier (e.g., glycerol, such as about 0.1% to about 50% glycerol).

[0149] Certain reagents can be used to dilute a sample or a portion of a sample. In some embodiments, one or more reagents are added to a sample or a portion of a sample in a ratio of sample to diluent to dilute the sample in a ratio of about 1:1 to about 1:2000 (e.g., about 1:1 to about 1:5, about 1:5 to about 1:10, about 1:10 to about 1:20, about 1:20 to about 1:50, about 1:50 to about 1:100, about 1:100 to about 1:300, about 1:300 to about 1:500, about 1:500 to about 1:1000, or about 1:1000 to about 1:2000).

[0150] The box includes one or more sensors connected to the chamber or channel fluid. The box can include 1, 2, 3, 4, 5 or more different sensors, and can include 1, 2, 3, 4, 5 or more redundant sensors of a given type of sensor. This article further describes in detail an exemplary sensor that can be used with the box. In short, the exemplary sensor includes an optical sensor and an impedance sensor, which can be configured to analyze biological samples. In some embodiments, the sensor is configured to detect proteins or electrolytes, or to measure protein concentrations or electrolyte concentrations in biological samples. In some embodiments, the sensor is a channel sensor (for example, a flow cytometer configured to count the number of cells in a biological sample, or a flow analyte sensor configured to detect or quantify the amount of an analyte such as a protein or enzyme). Flow cytometers may be able to distinguish different cell types (for example, red blood cells, white blood cells or platelets), and are configured to count one or more different types of cells.

[0151] The box can include a waste container, which is configured to receive the biological sample after being analyzed by one or more sensors of the biological sample. The waste container is connected to the main chamber, for example, by a waste channel fluid. The electrowetting electrode can be actuated by electrowetting to transport the analyzed biological sample to the waste container. Optionally, the waste channel and / or the waste container may include one or more electrowetting electrodes to control the delivery of the biological sample to the waste container. In some embodiments, once the biological sample has been transported to the waste container, it is retained in the waste container by hydrophobic action. The electrowetting electrodes adjacent to the waste container can be coated with a hydrophobic fluid contact layer, and when the electrowetting electrodes are not used, the fluid in the waste container remains there to avoid the hydrophobic fluid contact layer.

[0152] The box also includes a device interface, which is configured to receive power from the box interface on the device and communicate with it. The device interface includes a plurality of electrodes, which are electrically connected to the device electrodes, and the box and the device are engaged with each other. One or more sensors and electrowetting electrodes are electrically connected to the device interface through electrodes. This allows communication between the device and the box, and the device can operate the electrowetting electrodes to transport biological samples in the box, including flowing out and / or entering a waste container from a sample receiving port. The device can further operate the electrowetting electrodes to transport one or more reagents in the box and combine the biological sample with one or more reagents. The device can further operate one or more sensors in the box. One or more sensors can generate analysis data by analyzing the biological sample, and the biological sample is transmitted to the device through the device interface. In this way, the device can receive analysis data.

[0153] Optionally, the box may include one or more (e.g., 1, 2, 3, 4, 5 or more) optical windows that may be optically transparent on the top and / or bottom of the box. These optical windows may be used in combination with an impedance sensor or as an independent detector. Whether used independently or in combination, the reaction with the reagent may be detected by color change or fluorescence emission to selectively detect analyte or protein binding. In some embodiments, when used in combination with an impedance sensor, optical detection may be used to detect interfering substances not limited to lipids, bilirubin C, bilirubin F, chyle, hemolytic hemoglobin, glucose and / or alternatively detect sample quality to apply a correction factor for measurement. Sample quality inspection includes measurement of sample turbidity. If the box includes more than one optical window, the optical windows may have the same size or different sizes. The optical window is configured to allow light to pass through the sample, such as to capture an image of the sample or assay the sample using an optical microscope or spectroscopic examination (e.g., fluorescence, infrared (IR), Raman or UV / Vis absorption spectroscopic examination). The electrowetting electrode can deliver the biological sample or its part to the optical window, and the sample can be measured or the image of the sample can be captured. In some embodiments, the device of the operation box includes a light source and an optical detector aligned with the optical window when the box is engaged with the box. Then, the device can use the light source and the optical detector to measure the biological sample. In some embodiments, the device includes a camera aligned with the optical window of the box, and when the box is engaged with the device, the camera can image the biological sample through the optical window.

[0154] Figure 1A An exemplary box for analyzing biological samples is shown. The box includes a sample receiving port, which includes an inlet 102 that is fluidically connected to a sample receiving chamber 104 by a first sample port channel 106. The biological sample is deposited into the inlet 102 and travels into the receiving chamber 104 by capillary action through the first sample port channel 106. The sample receiving chamber 104 is fluidically connected to the main chamber 108 by the second sample port channel 110. Optionally, the box includes a filter (not shown) between the inlet 102 and the main chamber 108, which can be, for example, positioned in the first sample port channel 106 or the second sample port channel 110. If present, the filter can remove larger debris in the biological sample. The main chamber 108 includes a plurality of electrowetting electrodes 112, which continue to extend into the second sample port channel 110. The cartridge also includes a plurality of reagent containers 114a, 114b, 114c, and 114d, which are fluidly connected to the main chamber 108 via container channels 116a, 116b, 116c, and 116d. Figure 1AThe exemplary box shown in shows four reagent containers, but it should be understood that the box can include any number of containers, as determined by one or more tests performed using the box. Multiple electrowetting electrodes 112 extend into container channels 116a, 116b, 116c and 116d, and the electrowetting electrodes 112 can be operated to transport the reagents in container channels 116a, 116b, 116c and 116d to the mixing area 118 of the main chamber 108. The biological sample and the reagent are transported to the mixing area 118 and merged to form a processed biological sample. Optionally, the main chamber 108 of the box can include a filter. The electrowetting electrodes 112 can be used to transport the biological sample through the filter, for example, to separate the components of the biological sample (e.g., to separate serum from whole blood). Multiple electrowetting electrodes 112 can be operated to transport the processed biological sample or the portion of the processed biological sample to one or more sensors in the box. Optionally, the biological sample is analyzed by the sensor without being processed. Figure 1A The exemplary cartridge shown in FIG. 1 includes three impedance sensors 120 a, 120 b, and 120 c configured to measure the amount of protein in a biological sample. The impedance sensors may be of the same type (i.e., redundant impedance sensors performing the same assay) or of different types. Figure 1A The exemplary box shown in includes three impedance sensors, but it should be understood that the box can include any number of impedance sensors, which can depend on the number of tests or test redundancy of the particular box. The box shown also includes four channel sensors 122a, 122b, 122c and 122d, although other embodiments of the box may have additional or fewer channel sensors, which may be redundant or non-redundant. The channel sensor can be, for example, a flow cytometer, or can be configured to detect or quantify proteins. In the example shown, the plurality of electrowetting electrodes 112 include functionalized electrodes as part of the impedance sensors 120a, 120b and 120c. However, the plurality of electrowetting electrodes 112 do not extend into the channel sensors 122a, 122b, 122c and 122d, which use capillary action to pass the processed biological sample through the sensor. The box also includes a waste container 124 that is fluidly connected to the main chamber 108 via a waste channel 126. The plurality of electrowetting electrodes extend into a waste channel 126 and are operable to transport the processed biological sample to a waste container 124. The cartridge also includes a device interface 128 in electrical communication with the plurality of electrowetting electrodes 112 and sensors 120a, 120b, 120c, 122a, 122b, 122c, and 122d.

[0155] The cartridge optionally includes an optical window 130. The optical window includes a transparent window at the top of the cartridge and a transparent window at the bottom of the cartridge that allow light to pass through. The optical window 130 is fluidly connected to the main chamber 108, and the biological sample can be delivered to the optical window 130 using the electrowetting electrode 112. When the cartridge is engaged with the device ( Figure 1A When the optical window 130 is used as an optical device (not shown), the light source and the optical detector are positioned on opposite sides of the optical window 130, and the device can be used to measure a biological sample located in the optical window.

[0156] Figure 1B Another embodiment of a box with sensors having different orientations is shown. Although the size and / or spacing of the electrowetting electrodes can vary, the main chamber shows multiple electrowetting electrodes to demonstrate the array. The box includes a sample receiving port, which includes an inlet 132 fluidically connected to a sample receiving chamber 134 through a first sample port channel 136. The biological sample is deposited into the inlet 132 and travels into the receiving chamber 134 through the first sample port channel 136 by capillary action. The sample receiving chamber 134 is fluidly connected to the main chamber 138 through a second sample port channel 140. The receiving chamber 134 and the second sample port channel 140 include a first electrowetting electrode 142 and a second electrowetting electrode 144. The electrowetting electrodes are operated to controllably transfer the biological sample from the sample receiving chamber 134 through the second sample port channel 140 to the main chamber 138. Optionally, the cartridge includes a filter located between the inlet 132 and the main chamber 138, which filter can be located, for example, within the first sample port channel 136 or the second sample port channel 140. For example, if the filter is located within the second sample port channel 140, an electrowetting electrode can be used to transfer the biological sample through the filter to the main chamber 138. If the filter is present, larger debris in the biological sample can be removed.

[0157] The main chamber 138 includes a plurality of electrowetting electrodes that are electrically connected to a device interface 146 so that the device can be used to operate the electrowetting electrodes when the electrowetting electrodes are connected to the device. The cartridge also includes a plurality of reagent containers 148a, 148b, 148c, and 148d that are fluidly connected to the main chamber 138 via container channels 150a, 150b, 150c, and 150d. Although in Figure 1BThe exemplary box shown in Figure 1 shows four reagent containers, but it should be understood that the box can include any number of containers, as determined by one or more tests performed using the box. Reagent containers 148a, 148b, 148c and 148d and / or container channels 150a, 150b, 150c and 150d may include one or more electrowetting electrodes that can be used to transport the reagent contained in the reagent container to the main chamber. For example, reagent container 148a and reagent channel 150a may include a first electrowetting electrode 152a and a second electrowetting electrode 154a.

[0158] The biological sample and reagent are transported to the mixing area of ​​the main chamber 138 and combined to form the processed biological sample. Optionally, the main chamber 138 of the box may include a filter. The biological sample can be transported through the filter using an electrowetting electrode, for example to separate the components of the biological sample (e.g., to separate serum from whole blood). Multiple electrowetting electrodes can be operated to transport the processed biological sample or a portion of the processed biological sample to one or more sensors in the box. Optionally, the biological sample is analyzed by the sensor without being processed.

[0159] Figure 1B The exemplary cartridge shown in includes two impedance sensors 156a and 156b configured to measure the amount of protein within a biological sample. The impedance sensors can be configured to perform the same assay (i.e., redundant impedance sensors) or different assays. Figure 1B The exemplary box shown in includes two impedance sensors, but it should be understood that the box can include any number of impedance sensors, which can depend on the determination of the specific box or the number of determination redundancies. The box shown also includes two channel sensors 158a and 158b, although other embodiments of the box can have additional or fewer channel sensors, which can be redundant or non-redundant. The channel sensor can be, for example, a flow cytometer, or can be configured to detect or quantify protein. The electrodes from impedance sensors 156a and 156b and channel sensors 158a and 158b are electrically connected to the device interface 146. When the device operates the box, the device can transmit current and / or receive collected data through the electrically connected sensors.

[0160] Figure 1B The box shown in includes an optical window 160 and an optical window 162, which can have the same size or different sizes. The optical windows include a transparent window located at the top of the box and a transparent window located at the bottom of the box, which allows light to pass through. The optical windows are each fluidly connected to the main chamber 138 through a channel, and an electrowetting electrode can be used to transport a biological sample to the optical window. When the box engagement device ( Figure 1BWhen the optical window is used as an optical device (not shown), the light source and the optical detector are located on opposite sides of the optical window, and the device can be used to measure a biological sample located in the optical window.

[0161] Figure 1B The cartridge shown in FIG. 1 further includes a waste container 164 fluidly connected to the main chamber 138 via a waste channel 166. The waste container 164 and the waste channel 166 include electrowetting electrodes so that the biological sample being analyzed can be transferred from the main chamber 138 to the waste container.

[0162] The box can be configured to include various numbers of sensors and / or sensor types, depending on the assay or assay set that the box is used to perform. For example, the box can include one or more channel sensors (e.g., a flow cytometer or a channel sensor configured to detect an analyte such as a protein), an optical window, and / or an impedance sensor. The box can be configured to perform one or more assays or assay sets according to the configuration, reagents, and / or sensors included in the box. For example, the box can be configured to analyze one or more cell counts (e.g., red blood cell counts, white blood cell counts, and / or platelet counts), or detect or quantify proteins (or enzymes) or other analytes (e.g., electrolytes). In some embodiments, the box is configured to test a complete blood count (CBC) or a comprehensive metabolic set (CMP) of a biological sample. In some embodiments, the cartridge is configured to detect or quantify one or more of glucose, calcium, blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, carbon dioxide, total serum protein, albumin, total bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), red blood cells, white blood cells, platelets, hemoglobin, or hematocrit.

[0163] Device for operating box

[0164] Device is configured to dock with box as described herein and operate it, to form a system for analyzing biological sample.As discussed, the device can dock and operate different box types, and the box type is configured to perform different determinations or determination sets to biological sample.Described device includes box interface, and the device interface on the box engagement box is to power the box and communicate with the box.In this regard, the device is configured to power the box, to power one or more sensors and / or electrowetting electrodes, receive analysis data from one or more sensors in the box, and operate one or more sensors in the box.The device may include a processor (such as a microcontroller, a finite state machine, etc.) and a memory, which may be used for operating the device to control electrowetting electrodes or one or more sensors, and receive analysis data.In some embodiments, the device interface on the box is configured to be inserted into the box interface on the device, and in some embodiments, the box interface on the device is configured to be inserted into the device interface on the box.The device may be a handheld device, which may be powered by a battery.

[0165] The device can be configured to power and communicate with different types of cartridges to perform different analyses on biological samples. For example, the device can engage a first type of cartridge with a first sensor, analyze a first biological sample using the first type of cartridge, disengage the first type of cartridge, engage a second type of cartridge with a second sensor different from the first sensor, and analyze a second biological sample using the second type of cartridge. The device can be configured to power and operate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different types of cartridges.

[0166] In some embodiments, the device includes one or more processors and a non-transitory computer-readable storage medium storing one or more programs configured to be executed by one or more processors (e.g., microcontrollers). One or more programs may include instructions for operating multiple electrowetting electrodes in the box to transport biological samples and / or mix biological samples with one or more reagents. One or more programs may also include instructions for operating one or more sensors. As further described herein, some sensors can utilize static placement of biological samples in the sensor, and some sensors may require continuous flow of biological samples through the sensor. Therefore, in some embodiments, one or more programs include instructions for operating multiple electrowetting electrodes in the box to continuously transport biological samples through the sensor or statically place biological samples in the sensor.

[0167] The device includes hardware and circuits powered to different device components, and the device components include multiple electrowetting electrodes and one or more sensors. For example, the device may include one or more power circuits, and the power circuit provides energy to various box components to operate the box components. In some embodiments, the device is configured to use about 0.5 volt to about 1000 volts (for example, about 0.5 volt to about 1 volt, about 1 volt to about 5 volts, about 5 volts to about 10 volts, about 10 volts to about 20 volts, about 20 volts to about 30 volts, about 30 volts to about 50 volts, about 50 volts to about 100 volts, about 100 volts to about 250 volts, about 250 volts to about 500 volts, or 500 volts to about 1000 volts) to operate multiple electrowetting electrodes. Preferably, multiple electrowetting electrodes use about 50 volts or lower, or more preferably about 20 volts or lower to operate. Using multiple electrowetting electrodes to actuate the lower voltage of biological samples allows lower power to use, which can allow the device to be powered by batteries.

[0168] The device also includes electronic circuits and / or one or more programs to power, operate, and receive data from one or more sensors. For example, the device may include an integrated circuit (e.g., a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC)) for operating the sensor, which may include sending and / or receiving data using a data acquisition circuit (DAQ). The device may also include a digital signal processor (DSP) and / or a digital signal synthesizer (DSS).

[0169] The device may further include electronic circuitry to allow information display or data export and / or communication. For example, the device may include input / output ports (I / O ports) and / or wired or wireless communication means (such as WiFi or Bluetooth communication means).

[0170] The device can be docked with a cartridge as a system for analyzing biological samples. Figure 2A block diagram of an exemplary device engaged with a box is shown. The device includes a processor electrically connected to a memory, and the memory can store one or more programs for operating the device and / or the box. The device also includes a power circuit, which can include a battery or an electrical plug. The power circuit is electrically connected to the processor to provide power to the processor, and is electrically connected to the electrowetting electrode driver. The power circuit can provide different power supplies to the processor and the electrode driver. For example, depending on the configuration of the electrowetting electrode, the power circuit can provide 3.3 volts (or any other suitable voltage) to the processor and provide power between about 0.5 volts and about 1000 volts to the electrowetting electrode driver. The processor is configured to operate the electrowetting electrode driver, which is electrically connected to the electrowetting electrode through the box interface on the device and the device interface on the box. The processor can operate the electrowetting driver, which uses the electrowetting electrode to control the electrowetting drive of the fluid (e.g., biological sample and one or more reagents) passing through the box. The electrowetting driver operates the electrowetting electrode to transport the biological sample in the sample inlet to the mixing area of ​​the box. In addition, the electrowetting drive operates the electrowetting electrodes to transport the reagent in the reagent container to the mixing area to combine the reagent with the biological sample to form a processed biological sample. The electrowetting drive can then operate the electrowetting electrodes to transport the processed biological sample to the sensor (or multiple sensors, such as sensor 1 and sensor 2, if present on the cartridge) continuously or alternatively (e.g., by separating the sample), and then transport the processed biological sample to a waste container.

[0171] Continue to refer Figure 2, the processor of the device can be electrically connected to an integrated circuit (e.g., an FPGA) configured to operate one or more sensors (e.g., sensor 1 and sensor 2). Sensor 1 can be, for example, a flow cytometer configured to count the number of cells or the type of a certain cell in a biological sample, and sensor 2 can be, for example, an impedance sensor configured to quantify the concentration of a protein or analyte. The device may include a digital signal synthesizer (DSS) module that uses a direct digital synthesizer (DDS) and a digital-to-analog converter (DAC) to generate an analog signal (e.g., a multiplexed analog signal) and transmit it to one or more sensors. Alternatively, this multiplexed signal can also be generated using a direct analog synthesizer (DAS) that does not require a DAC. As shown, the DSS is embedded in the FPGA, but it can be considered that the DSS can be separated from the FPGA. In each of the above two schemes using DDS or DAS, the analog or digital signals need to be mixed after generation, which requires a separate mixing stage to generate the multiplexed analog signal. In a separate embodiment, an arbitrary waveform generator (AWG) is used to generate the signal, wherein a variable frequency clock is used to convert digital samples of a multiplexed signal stored in a memory or a lookup table (LUT) into an analog form. The data from the sensor is transmitted to a data acquisition circuit (DAQ), which can store the data in a memory (optionally embedded in an FPGA) and then retrieved by a processor (e.g., a digital signal processor (DSP)) for processing. The DSS can provide a reference signal to the DAQ circuit, which can be used to calibrate the signal received from the sensor. The DSP is optionally electrically connected to the device processor and can transmit the data to the device processor. The device processor can store the data in the device memory, or it can output the data through an input / output (I / O) port to display or transmit the data in other ways (e.g., through Bluetooth or a network (e.g., a WiFi connection)). Optionally, the DSP can communicate directly with the I / O port to transmit the data.

[0172] The biological sample enters the box through the sample inlet and optionally passes through a first filter (filter 1) before entering the main chamber of the box. An optional filter can be used to prevent large debris from entering the chamber. The main chamber includes an electrowetting electrode operated by the device, which can transport the biological sample to the mixing area of ​​the chamber. The device can also operate the electrowetting electrode to transport one or more reagents in a reagent container to mix with the biological sample in the mixing area. In some embodiments, a second filter (filter 2) can be included, and the electrowetting electrode operated by the device can transport the sample through the filter before the sample is transported to one of the one or more sensors. The optional second filter can be used to separate the components of the biological sample, such as separating serum or separating red blood cells and platelets from white blood cells.

[0173] sensor

[0174] The box can include one or more sensors that can analyze biological samples. In some embodiments, the electrowetting electrode is configured to transport the biological sample to one or more sensors for analyzing the biological sample. The sensor can transmit data to the device, and the device is electrically connected to the sensor by the device interface and the box interface. The one or more sensors in the box can vary with the type of box, depending on the required biological sample analysis. Exemplary sensors include channel sensors and impedance-based sensors. Described herein are sensors for detecting analytes (such as proteins, enzymes or electrolytes), measuring analyte concentrations (such as protein enzymes or electrolyte concentrations) or calculating cell counts or cell type counts. However, other sensors can be used together with the box described herein.

[0175] Analyte Sensors

[0176] In some embodiments, the cartridge includes an optical window or impedance sensor configured to detect and / or quantify an analyte, such as a protein, enzyme, or electrolyte.

[0177] The optical sensor that can be a part of the device engaged with the box can be aligned with the optical window of the box to analyze biological samples, such as detecting proteins or measuring protein concentrations. The optical sensor can use optical microscopy or spectroscopic examination (such as fluorescence, infrared (IR), Raman or UV / Vis absorption or any other suitable spectroscopic examination technology) to analyze biological samples to generate analytical data. The device can be used to analyze the data collected using spectroscopic examination to determine whether there is protein or protein concentration. In some embodiments, the box includes two or more optical windows, and at least one of the optical sensors can be used as a control. For example, a reagent without a biological sample can be analyzed by a control sensor, which can provide a baseline for calibration. Then, the device can calibrate the received data to measure accurate analyte concentrations.

[0178] In some embodiments, the sensor is an impedance sensor, which is configured to detect an analyte (e.g., protein, enzyme or electrolyte) or measure the amount of an analyte. Impedance sensors can work independently (or work together with other impedance sensors) or work in combination with one or more optical sensors. In some embodiments, when an optical sensor is used in combination with an electrical impedance-based sensor for analyte detection, the optical sensor can be used to detect interfering substances or detect a calibration factor for impedance measurement. The impedance sensor can include an electrode pair, wherein at least one of the two electrodes is functionalized by an affinity portion specifically bound to the target analyte. The electrode can be coated with an insulating layer (i.e., a dielectric layer), and the affinity portion can be functionalized on an insulating layer or an optional hydrophobic layer covering the insulating layer. The insulating layer preferably includes a high κ material (i.e., having a dielectric constant of about 3.9 or higher). In some embodiments, the insulating layer includes silicon dioxide, silicon nitride or silicon oxynitride or a combination thereof (e.g., mixed together or in the form of multiple separated layers). The properties (eg, thickness, dielectric constant, and / or hydrophobicity) of the insulating layer and / or the hydrophobic layer may be the same or similar to the properties of the insulating layer and the hydrophobic layer described herein.

[0179] The affinity moiety can be, for example, an antibody, an antibody fragment, or an aptamer (e.g., a DNA aptamer, an RNA aptamer, or an XNA aptamer). The biological sample can be delivered to the sensor, and the target protein binds to the affinity moiety. In some embodiments, the affinity moiety specifically binds hemoglobin.

[0180] In some embodiments, the biological sample is statically placed in the impedance sensor during impedance measurement, and in some embodiments, the biological sample flows through the sensor continuously during impedance measurement. The impedance variation between the two electrodes caused by the combination of the target molecule and the affinity molecule is related to the concentration of the analyte in the biological sample. In some embodiments, the box includes a reference sensor, which includes an electrode pair, which includes at least one functionalized electrode configured in a similar manner to the sensor for analyzing the biological sample. A control fluid such as a reagent without a biological sample can be used to detect a baseline impedance, which can be transmitted to the device and used to calibrate the sensor for analyzing the biological sample.

[0181] In some embodiments, impedance is measured at a sampling rate of about 10 kHz or more (e.g., about 20 kHz or more, about 50 kHz or more, about 100 kHz or more, about 200 kHz or more, about 300 kHz or more, about 400 kHz or more, about 500 kHz or more, about 1 MHz or more, about 5 MHz or more, about 10 MHz or more, about 25 MHz or more, about 50 MHz or more, about 75 MHz or more, about 100 MHz or more, about 125 MHz or more, or about 150 MHz or more). In some embodiments, the impedance is measured at a sampling rate of about 10 kHz to about 200 MHz (such as about 10 kHz to about 20 kHz, about 20 kHz to about 50 kHz, about 50 kHz to about 100 kHz, about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz, about 500 kHz to about 1 MHz, about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, about 4 MHz to about 5 MHz, about 5 MHz to about 10 MHz, about 10 MHz to about 25 MHz, about 25 MHz to about 50 MHz, about 50 MHz to about 75 MHz, about 75 MHz to about 100 MHz, about 100 MHz to about 125 MHz, about 125 MHz to about 150 MHz, or about 150 MHz to about 200 MHz). In some embodiments, the impedance is measured at a sampling rate of about 100 kHz to about 5 MHz. In some embodiments, impedance is measured at a sampling rate of about 125 MHz or higher.

[0182] In some embodiments, impedance is measured at an excitation frequency of about 1 Hz or more (e.g., 10 Hz) or 100 Hz or more or 1 kHz or more, 10 kHz or more (e.g., about 20 kHz or more, about 50 kHz or more, about 100 kHz or more, about 200 kHz or more, about 300 kHz or more, about 400 kHz or more, about 500 kHz or more, about 1 MHz or more, about 5 MHz or more, about 10 MHz or more, about 25 MHz or more, about 50 MHz or more, about 75 MHz or more, about 100 MHz or more, about 125 MHz or more, or about 150 MHz or more). In some embodiments, the impedance is measured at an excitation frequency of about 1 Hz to about 100 Hz, or about 100 Hz to about 1 kHz, or about 1 kHz to about 10 kHz, or about 10 kHz to about 200 MHz (e.g., about 10 kHz to about 20 kHz, about 20 kHz to about 50 kHz, about 50 kHz to about 100 kHz, about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz, about 500 kHz to about 1 MHz, about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, about 4 MHz to about 5 MHz, about 5 MHz to about 10 MHz, about 10 MHz to about 25 MHz, about 25 MHz to about 50 MHz, about 50 MHz to about 75 MHz, about 75 MHz to about 100 MHz, about 100 MHz to about 125 MHz, about 125 MHz to about 150 MHz, or about 150 MHz to about 200 MHz). In some embodiments, the impedance is measured at an applied excitation frequency of about 100 Hz to about 125 kHz. In some embodiments, the impedance is measured at a sampling rate of about 125 kHz or higher.

[0183] Figure 3An exemplary impedance sensor pair is shown, which is configured to measure the concentration of an analyte concentration such as a protein or enzyme concentration. The impedance sensor pair includes a test sensor and a control sensor. A biological sample is delivered to the test sensor, and a control buffer is delivered to the control sensor. Impedance is measured at the test sensor and the control sensor, and the impedance due to the analyte is the difference between the impedance measured at the test sensor and the control sensor. Both sensors include a first sensing electrode 302 on a bottom substrate 304 and a second sensing electrode 306 on a top substrate 308 opposite the first sensing electrode 302. The first sensing electrode 302 is coated with a first insulating layer 310, and the second sensing electrode 306 is coated with a second insulating layer 312. The first dielectric layer 310 is coated with a first hydrophobic layer 314, and the second dielectric layer 312 is coated with a second hydrophobic layer 316. The first hydrophobic layer 314 is functionalized with an affinity portion 318, such as an antibody, which specifically binds to an analyte 320 in the biological sample in the test sensor. The first hydrophobic layer 314 of the reference sensor is also functionalized by affinity part 318, but does not bind analyte because no biological sample is transported to the reference sensor. Although the second hydrophobic layer 316 can be functionalized in other embodiments, the second hydrophobic layer 316 in the sensor shown is not functionalized by affinity part. The biological sample is transported to the test sensor between the first sensing electrode 302 and the second sensing electrode 306, and the analyte impedance (Z2) can be measured. The control buffer is transported to the reference sensor, and the reference impedance (Z1) is measured. The difference between the analyte impedance (Z2) and the reference impedance (Z1) is related to the concentration of protein in the biological sample. The sensor is electrically connected to the device interface of the box. When the device interface of the box is engaged with the box interface on the device, the device can operate the impedance sensor to detect the change of impedance when the analyte is combined with the affinity molecule or the reference impedance. Then, the device can determine the analyte concentration according to the detected impedance.

[0184] Use multiple electrowetting electrodes to deliver biological samples to impedance sensors. In some embodiments, the first sensing electrode (or the second sensing electrode) is located between two electrowetting electrodes in the multiple electrowetting electrodes. In some embodiments, the first sensing electrode and / or the second sensing electrode are electrowetting electrodes within multiple electrowetting electrodes. In this configuration, multiple electrowetting electrodes can deliver biological samples to a position between the first sensing electrode and the second sensing electrode, and the first sensing electrode (or the second sensing electrode) can statically hold the biological sample in the sensor by activating the sensing electrode. The voltage switching circuit in the device or operated by the device is connected to the electrowetting electrodes, and a voltage can be applied to one of the electrodes to activate the electrodes, and the biological sample is attracted to position itself above the activation electrode. The voltage switching circuit is electrically connected to a switch, which is configured to alternately select between an impedance sensing circuit and an electrowetting electrode power supply circuit. The impedance sensing circuit is configured to be electrically connected to a processor in the device when the box is engaged with the device. The electrowetting power supply circuit is configured to be electrically connected to an electrowetting electrode driver in the device when the box is engaged with the device, and power can be supplied to the electrowetting electrodes.

[0185] Figure 4AAn exemplary impedance sensor integrated with multiple electrowetting electrodes is shown, which is configured to detect or measure an analyte (e.g., a protein, enzyme, or electrolyte). The multiple electrowetting electrodes include a first electrowetting electrode 410, a first sensing electrode 412, and a second electrowetting electrode 414. Opposite the first electrowetting electrode 410, the first sensing electrode 412, and the second electrowetting electrode 414 is a second sensing electrode 416, which also serves as a ground electrode for the multiple electrowetting electrodes. The first electrowetting electrode 410, the first sensing electrode 412, and the second electrowetting electrode 414 are coated with a first insulating layer 418, and the second sensing electrode 416 is covered with a second insulating layer 420. Although the second sensing electrode 416 is shown as a common electrode for electrodes 410, 412, and 414, it is conceivable that electrodes 410, 412, and 414 can be opposite to each electrode. At 422, at a position above the first sensing electrode 412, the first insulating layer 418 is functionalized with an affinity portion that specifically binds to a target analyte in a biological sample. The first electrowetting electrode 410, the first sensing electrode 412, and the second electrowetting electrode 414 are electrically connected to a voltage switching circuit 424. The voltage switching circuit 424 can selectively activate one of the first electrowetting electrode 410, the first sensing electrode 412, or the second electrowetting electrode 414, or deactivate any of them by applying a voltage to the second electrode. The voltage switching circuit 424 is electrically connected to a switch 426, which is configured to selectively connect the voltage switching circuit 424 to an impedance sensing circuit 428 or an electrowetting electrode power supply circuit 430. The impedance sensor may optionally include a first hydrophobic layer 432 covering the first insulating layer 418 and / or a second hydrophobic layer 434 covering the second insulating layer 420 (see Figure 4B ). At location 436 above the first sensing electrode 412, the first hydrophobic layer 432 is functionalized with an affinity moiety.

[0186] The biological sample can be delivered to the first electrowetting electrode 410 by applying a voltage to the first electrowetting electrode 410. The voltage selection circuit 424 selects the first electrowetting electrode 410, and the switch 426 electrically connects the voltage selection circuit 424 to the electrowetting electrode power supply circuit 430. In the case where the biological sample is located above the first electrowetting electrode 410, the voltage selection circuit 424 selects a voltage and applies it to the first sensing electrode 412, thereby deactivating the first electrowetting electrode 410. By selecting the first sensing electrode at 412, the biological sample is delivered to the first sensing electrode 412, where it can be statically maintained so that the target protein in the biological sample can bind to the affinity part at 422. The switch 426 can electrically connect the impedance sensing circuit 428 to the voltage selection circuit 424, thereby allowing the change in impedance to be detected. The change in impedance can be referenced to a similar reference sensor with a control solution instead of a biological sample. The concentration of the target analyte in the biological sample can then be determined based on the detected impedance. Once the analysis of the biological sample is completed, the switch 426 can be electrically connected to the electrowetting electrode power supply circuit 430 to the voltage selection circuit 424, and the voltage selection circuit 424 can select the second electrowetting electrode 414 and deactivate the first sensing electrode 412, thereby transporting the biological sample out of the impedance sensor. The biological sample can then be analyzed by a different sensor or transported to a waste container.

[0187] Impedance sensors for analyte detection or quantification can additionally or alternatively rely on indirect analyte detection or quantification methods. For indirect measurement, the analyte (e.g., protein, enzyme, or electrolyte) is bound to an affinity portion, washed with a wash buffer, and then bound to a second affinity portion that can produce ions or protons (e.g., by catalyzing a compound to produce hydrogen peroxide, which can form protons). The ions or protons can be detected by an ion-sensitive or pH-sensitive film, and the concentration of the ions or protons is proportional to the concentration of the analyte. Exemplary pH-sensitive layers can include hafnium oxide, aluminum oxide, iridium oxide, or chromium-tantalum oxide (CrO2 / Ta2O3). In some embodiments, a metal oxide semiconductor capacitor (MOSCap) sensor including a pH-sensitive layer or an ion-sensitive layer is used to detect ions or protons.

[0188] Exemplary impedance sensors that can be used to indirectly detect or quantify analytes are Figure 5A shown. Figure 5AThe impedance sensor shown includes a functionalized electrowetting electrode adjacent to a MOSCap sensor. An affinity portion is used to capture an analyte on the functionalized electrowetting electrode, and then a second affinity portion is bound to the analyte to produce ions or protons, which flow to the MOSCap sensor for detection. The electrowetting electrode includes a first electrowetting electrode 502 on a bottom substrate 504 and an optional second (grounded) electrowetting electrode 506 on a top substrate 508 opposite to the first electrode 502. The first electrowetting electrode 502 is coated with a first insulating layer 510, and the second electrowetting electrode 506 is coated with a second insulating layer 512. Optionally, a first hydrophobic layer 514 is coated on the first insulating layer 510 above the first electrowetting electrode 502, and a second hydrophobic layer 516 is coated on the second insulating layer 512 below the second electrowetting electrode 506. However, the first hydrophobic layer 514 and the second hydrophobic layer 516 do not extend into the MOSCap sensor. The first hydrophobic layer 514 (or the dielectric layer 510 in embodiments where the first hydrophobic layer is omitted) is functionalized with an affinity moiety 518. In some embodiments, the functionalized electrowetting electrode is configured to directly detect or quantify an analyte based on a change in impedance when the analyte binds to the affinity moiety.

[0189] The MOSCap sensor includes a first MOSCap electrode 520 on a first substrate 504 and a second MOSCap electrode 522 on a second substrate 508 opposite to the first MOSCap electrode 520. The first MOSCap electrode 520 is covered with a first semiconductor layer 524 (e.g., silicon, germanium, or a gallium compound, such as gallium arsenide or gallium nitride), and the second MOSCap electrode 522 is coated with a second semiconductor layer 526. In this embodiment, 504 and 508 can be different insulating or semiconductor materials from 524 and 526, or the same insulating or semiconductive material. Although it is considered that the insulating layer covering the semiconductor layer can be different from the insulating layer covering the electrowetting electrode, the first insulating layer 510 extends over the first semiconductor layer 524, and the second insulating layer 512 extends over the second semiconductor layer 526. The first detection layer 528 (e.g., a pH sensitive layer or an ion sensitive layer) covers the first insulating layer 510 in the MOSCap sensor, and the second detection layer 530 (e.g., a pH sensitive layer or an ion sensitive layer) covers the second insulating layer 512 in the MOSCap sensor.

[0190] Figure 5BA side view of a MOSCap sensor and an electrical model are shown. The MOSCap sensor includes a reference electrode 532 (which can be, for example, a silver or silver chloride electrode, or can be any other suitable material) and a counter electrode 534 (which can be, for example, gold or any suitable material). In some embodiments, the reference electrode 532 and the counter electrode 534 are positioned in line with the liquid flow (for example, the reference electrode 532 or the counter electrode 534 can be positioned between the first MOSCap electrode 520 and the first electrowetting electrode 502). In some embodiments, the reference electrode 532 and the counter electrode 534 are positioned adjacent to the liquid flow.

[0191] Figure 6 Shows the use of Figure 5A and 5B Direct and indirect analyte measurement of the sensor shown in . In step 602, the biological sample is transported to the functionalized electrowetting electrode by electrowetting actuation, and the analyte to be analyzed is bound to the affinity part attached to the hydrophobic layer (or insulating layer, if there is no hydrophobic layer). Optionally, once the analyte is bound, the biological sample is washed. Once the analyte is bound to the affinity part, the impedance can be measured to directly measure the analyte concentration, as described above. In step 604, the secondary affinity part conjugated with the signaling enzyme configured to produce protons is transported to the functionalized electrowetting electrode, and the analyte bound to the main affinity part attached to the electrowetting electrode is bound (through the insulating layer and / or the hydrophobic layer). In step 606, the reagent is transported to the electrowetting electrode, which can be catalyzed by the signal enzyme to produce hydrogen peroxide, which degrades to produce protons. In step 608, the protons flow to the MOSCap sensor, and the change in the pH sensitive layer responds to the proton modulated impedance. The modulated impedance is detected using a MOSCap sensor and is proportional to the concentration of the analyte in the biological sample.

[0192] In some embodiments, the impedance sensor is a MOSCap sensor in which one of the insulating layers (or the hydrophobic layer coating the insulating layer, if present) is functionalized with an affinity moiety. The opposing insulating layer is coated with a pH-sensitive or ion-sensitive layer. This configuration of the MOSCap sensor allows for direct analyte detection or concentration measurement (via the functionalized surface) and indirect analyte detection (via the pH-sensitive or ion-sensitive layer) in the same MOSCap sensor.

[0193] Channel Sensor

[0194] The box can additionally or alternatively include a channel sensor, which is configured to detect or quantify cells (or one or more types of cells) or analytes (such as proteins or enzymes) in a biological sample. In some embodiments, one or more channel sensors in the box include a flow cytometer. The flow cytometer can be configured to, for example, detect and distinguish multiple different types of cells, such as red blood cells, platelets and / or leukocytes (or one or more different types of leukocytes, such as eosinophils, basophils, neutrophils, monocytes and / or lymphocytes). In some embodiments, one or more channel sensors in the box include a flow analyte sensor. In some embodiments, one or more channel sensors can be configured as a flow cytometer and a flow analyte sensor, and optionally include multiple different electrode groups (each electrode group includes 2, 3 or more electrodes) configured for functionality (i.e., acting as a flow cytometer or a flow analyte sensor) in the same channel.

[0195] In some embodiments, the sample (e.g., blood sample) is not processed (e.g., by mixing with one or more reagents) before being analyzed by the channel sensor (e.g., to count and / or distinguish different cell types). For example, whole blood (possibly but not necessarily diluted or processed) can be passed through the channel sensor to count or distinguish cells in the whole blood sample. In some embodiments, the sample is processed before analysis, for example, by diluting the sample before analysis, marking certain cell types or cracking certain cell types (e.g., red blood cells). In some embodiments, the sample is processed by only diluting the sample before analysis (i.e., no labeling or cracking is required), and no additional processing steps are required before analysis. The sample can be processed, for example, by mixing the sample with one or more reagents.

[0196] Channel sensors are based on impedance sensing using the Coulter principle. Using the Coulter principle, a fluid-filled micropore or microchannel (or nanopore or nanochannel) with paired impedance sensing electrodes on either side of or inside the micropore or microchannel (or nanopore or nanochannel) is able to detect impedance modulation due to a single particle (such as a cell) passing through the pore or channel. The change in resistance or low-frequency impedance within the pore or channel is proportional to the particle size and depends on:

[0197]

[0198] Where R ch is the resistance of the channel or pore, ΔR chis the change in resistance of the channel or pore when a particle passes through the channel or pore, and d is the diameter of the particle. The fluctuation of the multiplexed impedance caused by particles (e.g., cells) flowing through the pore or channel depends on various properties of the particles, such as the size of the particles, the structure of the particles, the composition of the particles, or the surface properties of the particles. The impedance includes real components and imaginary components, and one or both of the components can be analyzed to determine the type of cell that generates the multiplexed impedance signal (i.e., an impedance signal of multiple frequencies). Alternatively, the amplitude component and phase component of the multiplexed impedance that can be measured and / or derived from the real component and / or the imaginary component can be analyzed to determine the type of cell that generates the multiplexed impedance signal (i.e., an impedance signal of multiple frequencies).

[0199] The channel sensor includes a channel including a micropore or microchannel (or a nanopore or nanochannel), through which a biological sample (processed in the box to dilute the sample and / or change the pH or electrolyte concentration of the surrounding buffer) can pass. The micropore or microchannel separates the first channel segment from the second channel segment, and the biological sample flows continuously through the entire channel. Although the electrowetting electrode can be used to introduce the biological sample into the channel sensor or withdraw the biological sample from the channel sensor, the biological sample passes through the sensor by capillary action rather than electrowetting actuation. The channel sensor also includes an impedance detection electrode group (e.g., an impedance detection electrode pair) configured to apply an electric current to the micropore or microchannel and detect the impedance in the micropore or microchannel. The impedance detection electrode is configured to contact the liquid flowing through the channel sensor. For example, the impedance detection electrode does not need to be separated from the sensor by an insulating layer (e.g., glass). In addition, as further discussed herein, the multivariate analysis of the detected electrical signal provides high-resolution detection of different cell types. Therefore, the channel sensor does not require a reference electrode.

[0200] Figure 7 An image of an exemplary channel of a channel sensor is shown having a first channel segment 702, a second channel segment 704, and a microchannel 706 between the first channel segment 702 and the second channel segment 704. The diameter of the microchannel is 50 μm.

[0201] In some embodiments, for analyte (e.g., protein) detection, the diameter of the micropore or microchannel (or nanopore or nanochannel) is about 10nm to about 1μm, or for flow cytometer, about 5μm to about 100μm. In some embodiments, for analyte (e.g., protein) detection, the diameter of the micropore or microchannel (or nanopore or nanochannel) is about 5nm to about 1μm. For example, in some embodiments, the diameter of the nanopore or nanochannel for analyte detection can be about 5nm to about 10nm, about 10nm to about 20nm, about 20nm to about 50nm, about 50nm to about 100nm, about 100nm to about 150nm or about 150nm to about 200nm. In some embodiments, the flow cytometer includes a micropore or microchannel with a diameter of about 5μm to about 100μm, for example, about 5μm to about 10μm, about 10μm to about 25μm, about 25μm to about 50μm or about 50μm to about 100μm. The size of the diameter can be selected based on the desired analysis, and different pore sizes can be used to more accurately count certain cell types. For example, it is preferred to use a channel sensor with a micropore or microchannel with a diameter of about 5 μm to about 25 μm to count platelets with an average diameter of about 1-3 μm, although larger diameters can be used for measurement. For leukocytes, the diameter of the micropore or microchannel is preferably about 40 μm to about 100 μm, such as about 50 μm.

[0202] The channel sensor may include two or more detection electrodes configured to detect impedance in a channel or hole (e.g., a microchannel or nanochannel, or a micropore or nanopore). In some embodiments, the detection electrode is configured to operate in pairs (e.g., a working electrode and a counter electrode). In some embodiments, the detection electrode is configured to operate as a triplet (e.g., a working electrode, a counter electrode, and a reference electrode). In some embodiments, the detection electrode is statically positioned in an electrode group (e.g., a group of two or three electrodes) for detecting impedance. In some embodiments, the electrodes are dynamically grouped into a group (e.g., by a device operating a box) to detect impedance. For example, the device can operate a switch that selects an electrode in a microchannel or nanochannel, and the selected electrode is used to detect impedance. Electrodes can be selected, for example, based on the spacing of the electrodes and the type of analyte or cell to be detected or measured.

[0203] The length of microchannel is usually greater than the diameter of microchannel. The detection electrode can be placed in the microchannel, outside the microchannel (that is, the microchannel is located in the space between the detection electrodes) or both. In some embodiments, the length of the microchannel is about 0.001mm or longer, for example, about 0.005mm or longer, about 0.01mm or longer, about 0.05mm or longer, about 0.1mm or longer, about 0.5mm or longer, about 1mm or longer, about 1.5mm or longer, about 2mm or longer, about 3mm or longer, or about 4mm or longer. In some embodiments, the length of the microchannel is about 5mm or shorter, for example, about 4mm or shorter, about 3mm or shorter, about 2mm or shorter, about 1.5mm or shorter, about 1mm or shorter, about 0.5mm or shorter, about 0.1mm or shorter, about 0.05mm or shorter, about 0.01mm or shorter, or about 0.005mm or shorter. In some embodiments, the length of the microchannel is about 0.001mm to about 5mm, for example, about 0.001mm to about 0.05mm, about 0.05mm to about 0.1mm, about 0.1mm to about 0.5mm, about 0.5mm to about 1mm, about 1mm to about 1.5mm, about 1.5mm to about 2mm, about 2mm to about 3mm, about 3mm to about 4mm or about 4mm to about 5mm in length. The channel (for example, microchannel or nanochannel) can include a plurality of detection electrodes (for example, two, three, four, five, six, seven, eight, nine, ten or more) along the length of the channel, and these electrodes can be divided into two or more groups. Two or more electrode groups can be used to obtain redundant or different impedance measurements. Redundant impedance measurements can be performed, for example, to improve detection by signal averaging. For example, different impedance measurements can depend on the different spacings of the electrodes, which can improve the detection sensitivity for measuring different cell types or sizes.

[0204] The spacing of the detection electrodes for measuring impedance can affect the detection sensitivity, which depends on the size of the measured particles. Generally, narrower electrodes are preferably used to measure smaller particles, while wider electrode spacing can be used for the measurement of larger particles. However, the spacing of the electrodes should not be too wide, so as to prevent multiple particles between the electrodes from measuring these same particles. For example, in some embodiments, the detection electrodes are spaced from about 5nm to about 100nm (for example, from about 5nm to about 10nm, from about 10nm to about 25nm, from about 25nm to about 50nm or from about 50nm to about 100nm) to measure or detect analytes (such as proteins, such as hemoglobin). In some embodiments, the detection electrodes are spaced from about 1μm to about 5μm (for example, from about 1μm to about 1.5μm, from about 1.5μm to about 2μm, from about 2μm to about 3μm, from about 3μm to about 4μm, or from about 4μm to about 5μm), for example, to detect or measure platelets. In some embodiments, the detection electrodes are spaced about 5 μm to about 10 μm (e.g., about 5 μm to about 6 μm, about 6 μm to about 7 μm, about 7 μm to about 8 μm, about 8 μm to about 9 μm, or about 9 μm to about 10 μm), for example, to detect or measure red blood cells. In some embodiments, the detection electrodes are spaced about 5 μm to about 25 μm (e.g., about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm), for example, to detect or measure white blood cells. Therefore, by including detection electrodes at different intervals within a channel, the same channel can be used to detect different analytes and / or cell types.

[0205] In some embodiments, the geometry of the channel of the sensor is uniform or approximately uniform (i.e., the diameter of the channel is constant or approximately constant). In some embodiments, the geometry of the channel of the sensor is tapered (i.e., the diameter of the channel increases or decreases along the length of the channel). The tapered geometry of the channel allows a single flow to be analyzed at different locations along the length of the channel using different channel diameters. In some embodiments, the geometry of the channel of the sensor includes one or more waves (i.e., an increase in diameter followed by a corresponding decrease in diameter), and optionally, one or more detection electrodes can be located within the waves.

[0206] The impedance sensing electrode pair may be located on either side of a micropore or microchannel (or nanopore or nanochannel), within a microchannel (or nanochannel) along the length of the microchannel (or nanochannel), or in a coplanar configuration within a micropore or microchannel (or nanopore or nanochannel). For example, Fig. 8AA top view and a side view of a channel sensor are shown having a first impedance detection electrode 802 in a first channel segment 804 and a second impedance detection electrode 806 in a second channel segment 808, with a microwell or microchannel 810 between the first channel segment 804 and the second channel segment 808. In the illustrated embodiment, the first impedance detection electrode 802 and the second impedance detection electrode 806 are both located on the lower surface of the channel sensor, but it is contemplated that the first impedance detection electrode 802 and the second impedance detection electrode 806 may be located on the upper surface of the channel sensor. Figure 8B A top view and a side view of a channel sensor are shown, which has a first impedance detection electrode 812 adjacent to a first channel segment 814 in a microchannel 820 and a second impedance detection electrode 816 adjacent to a second channel segment 818 in the microchannel. In the illustrated embodiment, the first impedance detection electrode 812 and the second impedance detection electrode 816 are both located on the lower surface of the microchannel, but it is contemplated that the first impedance detection electrode 812 and the second impedance detection electrode 816 can be located on the upper surface of the microchannel. Figure 8C In the embodiment, the channel sensor includes a first impedance detection electrode 822 and a second impedance detection electrode 824 located in a microchannel 826 (or a nanopore or a nanochannel), wherein the first impedance detection electrode 822 is located above the second impedance detection electrode 824 (i.e., the first impedance detection electrode 822 is on the upper surface, the second impedance detection electrode 824 is on the lower surface, and the first impedance detection electrode 822 is aligned with the second impedance detection electrode 824).

[0207] Fig.8D An exemplary channel sensor having two groups of detection electrodes within the same channel is shown. The sensor includes a first channel segment 828 and a second channel segment 830 separated by a channel (e.g., a microchannel) 832. The sensor includes a first group of detection electrodes 834 and a second group of detection electrodes 836 positioned along the length of the channel 832 and configured to measure the impedance within the channel 832. The first group of detection electrodes 834 includes a working electrode 838, a counter electrode 840, and an optional reference electrode 842 located between the working electrode 838 and the counter electrode 840. Similarly, the second group of detection electrodes 836 includes a working electrode 844, a counter electrode 846, and an optional reference electrode 848 located between the working electrode 844 and the counter electrode 846. The spacing between the working electrode 838 and the counter electrode 840 in the first group of detection electrodes 834 is different from (and in the example shown is greater than) the spacing between the working electrode 844 and the counter electrode 846 in the second group of detection electrodes 836. Therefore, in Fig.8D In the exemplary channel sensor shown in , the first set of detection electrodes 834 is optimized to detect larger particles than the second set of detection electrodes 836. Fig. 8E Shows something like Fig.8DAn exemplary channel sensor of the channel sensor shown in , except that the channel 832 includes waves 850, and the electrodes are respectively located within the waves. The diameter of the channel between the waves is narrower than the waves themselves.

[0208] Figure 8F An exemplary embodiment of a microchannel 852 is shown between a first channel segment 854 and a second channel segment 856, with a plurality of detection electrodes 858a-858p disposed along the length of the channel 852. In the illustrated embodiment, the electrodes 858a-858pp are evenly spaced, although in alternative embodiments, the electrodes are not evenly spaced. Electrode pairs can be dynamically selected to measure impedance.

[0209] In addition to the impedance detection electrode, the channel sensor may also optionally include one or more flow detection electrode pairs and / or one or more isolation electrodes. The flow detection electrode pair can be located on either side of the micropore or microchannel (or nanopore or nanochannel) and is configured to detect liquid flow. In some embodiments, the sensor includes a first flow detection electrode pair on the first side of the micropore or microchannel (or nanopore or nanochannel) (i.e., in the first channel segment), and a second flow detection electrode pair on the second side of the micropore or microchannel (or nanopore or nanochannel) (i.e., in the second channel segment). The flow detection electrode pair verifies whether the sensor is full of liquid, such as washing buffer, starting buffer or other control reagents by applying an electric current (e.g., DC current) and detecting a voltage change (or applying a voltage and detecting a current change). Once the device verifies that the sensor is full of liquid by electrically communicating with the flow detection electrode pair, the device can operate the electrowetting electrode in the box to transport the biological sample to the channel sensor.

[0210] A marker (e.g., bubble or low conductivity solution) can be introduced to separate the control reagent from the biological sample. Bubble markers can be introduced by interrupting the liquid flow from the channel sensor entrance, such as by operating the electrowetting electrode to stop the liquid flow. Capillary action causes the liquid in the channel to continue to flow through the channel sensor and generate bubbles. If the marker is a bubble, a bubble trap can be included in the channel sensor to prevent bubbles from passing through the hole or channel. The bubble trap may include a selective membrane that allows air diffusion but does not allow liquid diffusion. In some embodiments, the marker is an immiscible liquid, such as glycerol or oil (e.g., silicone oil, vegetable oil or olive oil). Liquid can then be introduced into the channel sensor so that the bubble is located between the two fluid streams.

[0211] The detection of the marker then confirms that the biological sample is introduced into the channel sensor and data acquisition is started. The marker can also be used to dynamically determine the flow rate of the liquid in the sensor, for example, based on the volume of the marker and the start and end time points of the marker passing through the flow detection electrode. Knowing the flow rate of the liquid in the channel sensor allows the concentration of particles (such as cells) in the biological sample to be quantified. The particle concentration is determined by the number of particles passing through the unit time divided by the flow rate. The particle concentration can also or alternatively be obtained by dividing the total number of particles by the volume through the channel sensor. The peak width of the detected impedance signal is also a function of the particle velocity through the channel, and the particle velocity through the channel can be calculated from the flow rate and measured from the pulse width. The particle velocity allows the translocation path of the particle through the channel to be determined based on the pulse width variation in the detected signal. This allows error correction to be applied to the amplitude of the measured signal, thereby allowing the signal intensity of the particles passing through the channel away from the central axis of the channel to be corrected. For smaller particles through the channel, the change will be more obvious. For example, if a 1-5 μm particle passes through a 50 μm channel, the particle may take multiple paths through the channel, either the center of the channel (indicating the true amplitude) or the edge, which results in a larger signal amplitude. Alternatively, the particle or cell may take a path between the center and the edge of the channel, resulting in an increase in amplitude between the two signal intensities.

[0212] The isolation electrode can be located near the inlet or outlet of the channel sensor and serves as a ground electrode. The ground (isolation) electrode isolates the channel sensor from electrical activity elsewhere in the cartridge, such as that resulting from the operation of the electrowetting electrode. Thus, the isolation electrode minimizes electrical noise within the channel sensor.

[0213] Fig. 9 A top view and a side view of a channel sensor including a pair of impedance detection electrodes, two pairs of flow detection electrodes, and two isolation electrodes are shown. A first set of electrowetting electrodes 902 is adjacent to the inlet of the sensor so that a control agent or a biological sample can be transported into the sensor, and a second set of electrowetting electrodes 904 is adjacent to the outlet of the sensor so that a control agent or a biological sample can be transported away from the sensor. Within the sensor, the control agent or the biological sample is transported by capillary actuation. The sensor includes a micropore 906 having a first impedance detection electrode 908 and a second impedance detection electrode 910 on opposite sides of the micropore 906. Although Fig. 9 The embodiment shown in shows impedance sensing electrodes on either side of the microwell, other configurations are contemplated (see Figures 8A-8C). The sensor also includes a first pair of flow detection electrodes 912 and 914 and a second pair of flow detection electrodes 916 and 918 on either side of the micropore 906. The flow detection electrodes are positioned further away from the micropore than the impedance detection electrodes. The sensor also includes a first isolation electrode 920 near the inlet of the sensor and a second isolation electrode 922 near the outlet of the sensor.

[0214] Fig.10 Shows Fig. 9 The beginning of the operation of the channel sensor shown, the curve below represents the DC voltage detected by the first pair of flow detection electrodes 912 and 914. At the beginning, the electrowetting electrode transports the fluid (e.g., a control reagent) into the channel. Since no liquid is in contact with the electrodes, the voltage detected by the flow detection electrode in the first step is minimal. When the liquid enters the channel in the second step, the electrowetting transport of the liquid to the sensor will produce a capillary flow through the sensor, thereby increasing the voltage detected by the flow detection electrode until the sensor is filled in the third step. A marker (e.g., an air bubble or a low conductivity buffer relative to the control reagent) is introduced into the sensor, and the control reagent is separated from the biological sample. Once the marker reaches the flow detection electrode in the fourth step, the voltage detected by the flow detection electrode drops. The voltage drop can signal the device to indicate that the biological sample is introduced into the sensor and data collection can be initiated.

[0215] The channel sensor can be reused in multiple analysis cycles, for example to measure analytes or cells under different conditions (e.g., different pH or electrolyte concentrations) or at different dilutions. Different pH values ​​or electrolyte concentrations of reagents surrounding cells can modulate the surface charge of the cells, and different surface charge changes may be associated with different cell types. The biological sample can be divided into multiple subsamples, which can be mixed (or serially diluted) with different reagents or different amounts of reagents within the box, and one or more processed subsamples can be cycled through the channel sensor. The subsamples are separated by a control buffer, which itself can be separated from the subsamples by a marker (e.g., an air bubble or a low conductivity buffer).

[0216] The biological sample can be diluted in the box by mixing the biological sample with one or more reagents. In some embodiments, before being measured by the channel sensor, the biological sample is diluted to one or more different concentrations, for example, to about 0.00001X to less than 1X (e.g., about 0.00001X to about 0.0001X, about 0.0001X to about 0.001X, about 0.001X to about 0.01X, about 0.01X to about 0.1X, or about 0.1X to about less than 1X). The pH of the separated biological sample can be adjusted within the box so that the biological sample can be measured at one or more pH levels, such as between 3.0 and about 9.0 (e.g., between about 3.0 and about 3.5, between about 3.5 and about 4.0, between about 4.0 and about 4.5, between about 4.5 and about 5.0, between about 5.0 and about 5.5, between about 5.5 and about 6.0, between about 6.0 and about 6.5, between about 6.5 and about 7.0, between about 7.0 and about 7.5, between about 7.5 and about 8.0, between about 8.0 and about 8.5, and / or between about 8.5 and about 9.0). In some embodiments, the pH of the biological sample measured is 7.4. The box can include several different reagents to adjust the pH of the biological sample, or can include two reagents that are mixed with the biological sample in a certain ratio to obtain the desired pH. In some embodiments, a biological sample is split and processed to obtain 1 to 5 different sample concentrations at 1 to 10 different pH levels, for a total of about 1 to about 50 different biological subsamples.

[0217] Fig.11 It is shown how a marker can be used as a trigger for data collection using the sensor, and the cycle can be repeated for different biological subsamples. Before fluid is introduced into the channel sensor ("no flow"), the liquid flow and flow rate are zero, and the voltage measured by the flow detection electrodes is small or no voltage. When a control reagent is introduced into the channel sensor, the rate of liquid flowing through the sensor increases, and as the control reagent flows through the flow detection electrode pair, the voltage measured by the flow detection electrode pair near the sensor inlet increases. Fig.11As shown, although it is conceivable that the trigger for data acquisition can be the detection of the first marker, the increase in voltage detected by the flow detection electrode triggers data acquisition. The control reagent is separated from the biological sample (or biological sub-sample, if the sensor is used for multiple cycles to analyze multiple processed sub-samples) by the marker. The marker flows through the sensor and the liquid drips (assuming there is a bubble marker). The marker across the flow detection electrode causes the measured voltage to drop, as shown in Figure 1, until the biological sample enters the sensor. The flow detection electrode detects the entry of the biological sample, marked by the increase in measured voltage. The biological sample is followed by a second control reagent (which may be the same or different from the first control reagent), which can be separated from the biological sample by a second marker. The second marker flows through the channel sensor, and the liquid drips (assuming that the second marker is a bubble). The flow detection electrode also detects the marker by measuring the drop in voltage. The voltage drop caused by the marker or the detection of the marker passing can optionally trigger the termination of data collection. When the second control reagent enters the sensor, the voltage measured by the flow detection electrode increases until the next marker that separates the second control reagent from the next round of biological samples is detected. The cycle can then continue to repeat in this pattern until the desired number of cycles is reached.

[0218] Fig.12An exemplary method for measuring a biological sample using a channel sensor in a box is shown. The device can determine the type of test to be performed based on the box inserted into the device or manually inputting the test type into the device. At the beginning of the test, the biological sample is added to the input port and then the sample is transferred to the mixing area by electrowetting drive. Based on the test performed on the biological sample, the biological sample is processed to obtain a predetermined number of different pH conditions and the biological sample is divided into a plurality of different pH conditions. The pH regulator is prepared in the box, for example, by mixing a high pH component and a low pH component in a predetermined ratio to obtain a desired number (x) of different pH regulators at a desired pH level. Then the subsample is mixed with the pH regulator (i.e., subsample [n] is mixed with pH regulator [n], subsample [n+1] is mixed with pH regulator [n+1], etc.). In some embodiments, the subsample is analyzed at isotonic pH (7.4) and the pH of the subsample is not adjusted. Then the subsample is diluted (or serially diluted) to the desired concentration. In addition, a control reagent is prepared, which is similar to the conditions of the treated biological subsample without containing the biological sample (cells, analytes, etc.). A control reagent is prepared for each different pH condition. The first control reagent (i.e., the control reagent for subsample [n]) is delivered to the channel sensor, followed by a marker (e.g., air bubbles or a low conductivity fluid). The first subsample (i.e., the subsample [n] of the desired concentration) is then delivered to the channel sensor, followed by another marker and the next control reagent (if the next subsample has the same reagent conditions and has a different dilution factor, the same control reagent is delivered). Another marker is introduced into the channel sensor, followed by the next subsample. This cycle is repeated until the subsample is analyzed by the channel sensor.

[0219] In some embodiments, the channel sensor described herein is a flow cytometer configured to detect a large number of cells or a plurality of one or more types of cells (e.g., white blood cells, platelets, or red blood cells). The electrical model of cells flowing through a hole or channel is Fig.13 In this model, C p Refers to the capacitance of the cell membrane, cytoplasm and nucleus; R p refers to the resistance from cells and indicates the volume of cells; C f and R f C refers to the capacitance and resistance of the fluid surrounding the cell, respectively, which can vary based on the electrolyte concentration or pH of the fluid; dl It refers to double layer capacitance.

[0220] The flow cytometer can work by applying a multi-channel current and detecting the multiplexed impedance across the micropore or microchannel when the cell flows through the micropore or microchannel. The impedance at any given current frequency in the multi-channel current may include a real component and an imaginary component, and one or two impedance components may be analyzed at different frequencies. Alternatively, the impedance at any given current frequency in the multi-channel current may include an amplitude component and a phase component, and one or two of the impedance components may be analyzed at different frequencies. The flow cytometer channel sensor may be configured to distinguish different types of cells in a biological sample. For example, the impedance to the multi-channel current may be used to distinguish different types of cells in a biological sample, such as white blood cells, red blood cells, and platelets. In some embodiments, the flow cytometer channel sensor may distinguish eosinophils, basophils, neutrophils, monocytes, and lymphocytes. Different types of cells may be distinguished based on the unique impedance characteristics of each cell type, which may include real impedance components and / or imaginary impedance components at multiple different current frequencies (e.g., 3, 4, 5, 6, 7, 8 or more different current frequencies). Alternatively, different types of cells can be distinguished based on a unique impedance signature for each cell type, which can include an amplitude impedance component and a phase impedance component at multiple different current frequencies (e.g., 3, 4, 5, 6, 7, 8 or more different current frequencies) measured directly or derived from real and imaginary impedance components. In some embodiments, the same electrode pair or different electrode pairs can be used to apply current and measure impedance. Using the flow cytometer design described herein, the box does not need to separate different types of cells to obtain a cell count for each cell type.

[0221] The impedance sensing electrode pair is configured to simultaneously provide multiple currents through the micropore or microchannel, and measure the voltage when the biological sample flows through the sensor to determine the impedance over a period of time. The multiple current includes (1) a DC component or a low-frequency AC current, and (2) multiple (e.g., 2, 3, 4, 5, 6, 7, 8 or more) AC components at different frequencies. Preferably, the multiple current includes at least three AC components. In another preferred embodiment, the multiple current includes at least five AC components.

[0222] The impedance detected at one or more frequencies can be used to distinguish the type of cells (e.g., white blood cells, red blood cells, or platelets, or a specific type of white blood cells, such as eosinophils, basophils, neutrophils, monocytes, or lymphocytes) passing through the micropore or microchannel. Different particle sizes, the presence or absence of a nucleus, and other cell characteristics affect the impedance at different current components. DC or low-frequency current components can be used to determine different cell sizes. DC current or low-frequency current minimizes or eliminates the effect of voltage drop at the electrode electrolyte (from the surrounding fluid) interface, thereby minimizing the double-layer capacitance in the electrical module. The resulting effect of impedance is related to the size of the particle (e.g., cell). Medium-frequency alternating current involves the portion of the cell capacitance affected by the cytoplasm and cell membrane. High-frequency alternating current components can be used to characterize the cytoplasm and nucleus (if present), while ultra-high-frequency alternating current is related to the capacitance of the nucleus (if present). Multiple currents can include other frequencies to better distinguish different cell types.

[0223] In some embodiments, the AC component includes (1) a direct current or low frequency AC current of about 1 kHz to about 100 kHz (e.g., about 1 kHz to about 10 kHz, about 10 kHz to about 20 kHz, about 20 kHz to about 30 kHz, about 30 kHz to about 40 kHz, about 40 kHz to about 50 kHz, about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, or about 90 kHz to about 100 kHz), (2) a direct current or low frequency AC current of about 100 kHz to about 700 kHz (e.g., about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz, about 500 kHz to about 600 kHz, about 600 kHz to about 700 kHz), or (3) a direct current or low frequency AC current of about 100 kHz to about 700 kHz (e.g., about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz, about 500 kHz to about 600 kHz, about 600 kHz to about 700 kHz). kHz), (3) a medium frequency alternating current of about 700 kHz to about 5 MHz (e.g., about 700 kHz to about 800 kHz, about 800 kHz to about 900 kHz, or about 900 kHz to about 1000 kHz, or about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, or about 4 MHz to about 5 MHz), and (4) an ultra-high frequency alternating current of 5 MHz or higher (e.g., about 5 MHz to 10 MHz, about 10 MHz to about 20 MHz, about 20 MHz to about 30 MHz, about 30 MHz to about 40 MHz, about 40 MHz to about 50 MHz, about 50 MHz to about 60 MHz, about 60 MHz to about 70 MHz, about 70 MHz to about 80 MHz, about 80 MHz to about 90 MHz, or about 90 MHz to about 100 MHz).In another embodiment, the multiplexed current comprises (1) a direct current or an alternating current of about 1 kHz to about 100 kHz (e.g., about 1 kHz to about 10 kHz, about 10 kHz to about 20 kHz, about 20 kHz to about 30 kHz, about 30 kHz to about 40 kHz, about 40 kHz to about 50 kHz, about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, about 90 kHz to about 100 kHz), (2) a direct current or an alternating current of about 50 kHz to about 25 (3) an alternating current of about 250 kHz to about 700 kHz (e.g., about 250 kHz to about 300 kHz, about 300 kHz to about 350 kHz, about 350 kHz to about 400 kHz, about 400 kHz to about 500 kHz), (4) an alternating current of about 150 kHz to about 200 kHz, or about 200 kHz to about 250 kHz, (5) an alternating current of about 250 kHz to about 700 kHz (e.g., about 250 kHz to about 300 kHz, about 300 kHz to about 350 kHz, about 350 kHz to about 400 kHz, about 400 kHz to about 500 kHz), (6) an alternating current of about 150 kHz to about 200 kHz, or about 200 kHz to about 250 kHz. (4) an alternating current of about 700 kHz to about 5 MHz (e.g., about 700 kHz to about 800 kHz, about 800 kHz to about 900 kHz, or about 900 kHz to about 1 MHz, or about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, or about 4 MHz to about 5 MHz); (5) an alternating current of about 5 MHz to about 20 MHz (e.g., about 5 MHz to about 10 MHz, about 10 In some embodiments, the plurality of AC components include an AC of about 50 kHz to about 250 kHz, a second AC of about 250 kHz to about 700 kHz, a third AC of about 700 kHz to about 5 MHz, a fourth AC of about 5 MHz to about 20 MHz, and a fifth AC of about 20 MHz to about 150 MHz. In some embodiments, the AC also includes an additional AC of about 100 kHz or less or about 50 kHz or less, which is useful for determining the size of cells.The frequency of the AC component can be selected based on salt concentration, channel or pore size, electrode size, spacing or material, or the specific cells to be counted.

[0224] In some embodiments, the multiple currents are generated as analog current components and mixed in real time using the device before being transmitted to the impedance detection electrodes in the box. For example, the device can include a digital signal synthesizer (DSS) module that uses a direct digital synthesizer (DDS) and a digital-to-analog converter (DAC) to generate and send analog signals (e.g., multiplexed analog signals). Alternatively, in some embodiments, an arbitrary waveform generator (AWG) is used to generate the multiple current signals, wherein a variable frequency clock is used to convert digital samples of the multiplexed signals stored in a memory or a lookup table (LUT) into analog form.

[0225] When the impedance is measured by the impedance detection electrode, the cells in the biological sample pass through the micropore or microchannel. In some embodiments, the impedance is measured at a sampling rate of about 10kHz or higher (e.g., about 20kHz or higher, about 50kHz or higher, about 100kHz or higher, about 200kHz or higher, about 300kHz or higher, about 400kHz or higher, about 500kHz or higher, about 1MHz or higher, about 5MHz or higher, about 10MHz or higher, about 25MHz or higher, about 50MHz or higher, about 75MHz or higher, about 100MHz or higher, about 125MHz or higher, or about 150MHz or higher). In some embodiments, impedance is measured at a sampling rate of about 10 kHz to about 200 MHz, such as about 10 kHz to about 20 kHz, about 20 kHz to about 50 kHz, about 50 kHz to about 100 kHz, about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz, about 500 kHz to about 1 MHz, about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, about 4 MHz to about 5 MHz, about 5 MHz to about 10 MHz, about 10 MHz to about 25 MHz, about 25 MHz to about 50 MHz, about 50 MHz to about 75 MHz, about 75 MHz to about 100 MHz, about 100 MHz to about 125 MHz, about 125 MHz to about 150 MHz, or about 150 MHz to about 200 MHz. Alternatively, in some embodiments, such as when an analog-to-digital converter (ADC) is used, the measured signal is downsampled, thereby shifting the frequency response to a lower frequency and allowing the signal to be digitized at a lower sampling rate. In some embodiments, the impedance is measured at a sampling rate of about 100 kHz to about 5 MHz. In some embodiments, the impedance is measured at a sampling rate of about 125 MHz or higher.

[0226] Fig.14The relative impedance change as a function of time measured by an impedance detection electrode at a single frequency (30kHz) for about 10 μm Chlorella cells (bacteria-free, available from Carolina Biological Supply, North Carolina, USA) is shown. Each downward peak corresponds to a particle passing through a microchannel, and various components of the impedance peak, such as peak height, peak width, peak area, and / or half-width peak height, can be measured. Multivariate patterns are constructed from multiple current components to distinguish different types of cells. Impedance changes as a function of baseline impedance are measured, and one or more components of the impedance change can be used to describe the detected cells. Such components include, but are not limited to, one or more of impedance peak height, impedance peak width, impedance peak area, and impedance half-width height, which indicate cell size, surface charge, cell shape, and other distinguishing features of cells. As further discussed herein, biological samples can also be measured at different concentrations or at different pH to further distinguish cell types. Since the impedance components are affected by the isoelectric point and surface charge of the cells, different cell types can be distinguished by multivariate patterns. Therefore, the unique multivariate pattern for different cell types can include impedance peak height, impedance peak width, impedance peak area and / or impedance half width height, which can be determined at one or more pH levels and / or one or more electrolyte concentrations. In some embodiments, the multivariate pattern can include real and imaginary components of impedance, for example, the amplitude from the real component and the amplitude from the imaginary component. In some embodiments, the multivariate pattern can include the amplitude component and phase component of impedance, for example, the amplitude from the amplitude component and the amplitude from the phase component. The amplitude component and phase component of impedance can be measured directly, or can be derived from the real component and the imaginary component. Clustering algorithms can be used to create patterns for various types of cells based on multivariate patterns collected at various frequencies. This can also be used to distinguish normal cell populations from abnormal cell populations.

[0227] By analyzing the multivariate pattern of detected impedance rather than just the impedance amplitude, the differentiation of different cell types is more accurate. For example, the channel sensor does not need to include a reference electrode to control impedance changes. In addition, the flow sensor can be used to analyze cell counts and / or analyte concentrations using whole blood biological samples rather than for analyzing isolated components of biological samples (e.g., serum, platelets, etc.) or labeled cells.

[0228] In some embodiments of the flow cytometer channel sensor, the real component and the imaginary component of the impedance signal are analyzed. For example, the multivariate mode of the real component of the multiplexed impedance and the multivariate mode of the imaginary component can be used to distinguish cell types. In some embodiments, the amplitude of the real component of the multiplexed impedance and the amplitude of the imaginary component are used to distinguish different cell types. In some embodiments of the flow cytometer channel sensor, the amplitude component and the phase component of the impedance signal are analyzed. For example, the multivariate mode of the amplitude component of the multiplexed impedance and the multivariate mode of the phase component can be used to distinguish cell types. In some embodiments, the amplitude of the amplitude component of the multiplexed impedance and the amplitude of the phase component are used to distinguish different cell types. The number of different current frequencies used for multiplexing impedance can be 3, 4, 5, 6, 7, 8 or more different current frequencies.

[0229] A filter (e.g., a bandpass filter) can be used to filter and demodulate the multiplexed impedance detected from the flow cytometer channel sensor to isolate each frequency. For example, this can be done by means of an operating box. The processor can analyze the multiplexed impedance signal for each cell passing through the flow cytometer channel sensor, and mark or count individual cells. For example, this can be done using a trained machine learning model. The machine learning model can be trained using known impedance features of various cell types. Exemplary machine learning models include naive Bayes classifiers, logistic regression, decision tree models, gradient boosting trees, support vector machines, neural networks, and other deep learning algorithms. In some embodiments, supervised or semi-supervised methods are used to train the machine learning model.

[0230] In one embodiment, unlabeled impedance features of particles or cells from a sample (e.g., whole blood) are used to refine the model by semi-supervised learning, which can include applying self-training to generate clusters of different particle / cell types characterizing the sample. The generated data set is compared with a benchmark data set. Errors in the unlabeled data set are used to calculate calibration weights for each cell type. In addition to repeated testing of single cell types from different sources, baseline weights can also be adjusted using mixed cell types of known types and whole blood samples to correct the fit of the model cluster to each cell type, while taking into account the patient's demographic information, such as age, gender, race, etc. For example, the model can establish clusters that distinguish white blood cells, red blood cells, and / or platelets. In some embodiments, clusters are distinguished within white blood cells, such as to distinguish two or more of neutrophils, basophils, eosinophils, monocytes, and / or lymphocytes. Clustering can be verified, for example, by the goodness of fit of one or more generative models (e.g., Gaussian mixture models). When total counting is performed using known methods (e.g., commercial hematology analyzers), another calibration factor can correct the counting error.

[0231] The box may additionally or alternatively include one or more channel sensors configured to measure the concentration of an analyte (e.g., a protein or enzyme). Although a similar Coulter principle can be applied, due to the smaller size of the particles, the aperture of the channel sensor used to measure the concentration of the analyte is generally smaller than the aperture used by the flow cytometer. Since the cells in the biological sample may clog the nanopore or nanochannel, the biological sample or subsample may be filtered before being transported to the channel sensor. Before the biological sample (or subsample) is introduced into the channel sensor, the biological sample (or subsample) is mixed with a reagent (which may include, for example, an antibody, an antibody fragment, or an aptamer) containing a charged affinity portion, which can specifically bind to the target analyte. In some embodiments, the affinity molecule is a multivalent affinity molecule (e.g., divalent, trivalent, or tetravalent). When passing through a nanopore or nanochannel, the charged affinity portion bound to the target analyte increases the electric field modulation compared to the target analyte, thereby increasing the impedance signal.

[0232] In some embodiments, a biological sample is analyzed using a channel sensor configured to measure the concentration of an analyte, and an affinity molecule (which may include, for example, an aptamer or an antibody or a fragment thereof) is bound to a multivalent tag, such as a protein or a nanoparticle. In some embodiments, the multivalent tag is a divalent tag, a trivalent tag, or a tetravalent tag. For example, in some embodiments, the multivalent tag is avidin or an avidin derivative (such as neutravidin or streptavidin), which can be combined with a biotin moiety fused to an affinity molecule. For example, in some embodiments, the affinity molecule is an aptamer with a biotin moiety, which is (optionally, via a linker) connected to the 5' or 3' end of the aptamer, and the multivalent tag is avidin. By using a multivalent tag, the impedance signal increases compared to using an affinity molecule alone.

[0233] The affinity part that is bound to the target analyte can be distinguished from the unbound affinity part based on the change of impedance, and the number of the bound affinity parts can be used to determine the concentration of the target analyte in the biological sample. In some embodiments, the change of impedance amplitude is used to distinguish the bound affinity part and the unbound affinity part. In some embodiments, a multivariate pattern is constructed from multiple current components to distinguish the bound and unbound affinity parts. The impedance change as a function of the baseline impedance is measured, and one or more components of the impedance change can be used to describe the state (bound or unbound) of the affinity part. Such components include but are not limited to one or more of the impedance peak height, impedance peak width, impedance peak area and impedance half-width high.

[0234] Fig.15AThe binding of a target analyte to a tetravalent affinity molecule is shown. A biological sample (which can be processed and / or filtered in a box by mixing the biological sample with one or more reagents) is mixed with a reagent comprising a tetravalent affinity molecule. The tetravalent affinity molecule includes a tetravalent tag (e.g., avidin or avidin derivative) that is bound to four aptamer molecules. The aptamer molecule is bound to the biotin portion through a linker sequence, and the anchor portion (e.g., biotin) is bound to the tetravalent tag. When the affinity molecule is mixed with the target analyte, a maximum of four target analyte molecules bind to the tetravalent affinity molecule.

[0235] Fig. 15B The impedance change (upper figure) caused by the affinity part that is not bound to the target analyte when the affinity part passes through the nanopore or nanochannel of the channel sensor and the impedance change (lower figure) caused by the affinity part combined with the target analyte are shown. As shown in step 2, the impedance signal can be detected in both cases, but when the affinity part is combined with the target analyte, the amplitude of the impedance signal is larger. Although the figure only shows the change of impedance amplitude, multivariate mode difference can be detected between these two cases. In some embodiments, the impedance amplitude is analyzed to determine the analyte concentration in the biological sample. In some embodiments, the multivariate mode is analyzed to determine the analyte concentration in the biological sample. The multivariate mode can include the characteristics of the impedance peak detected, such as impedance peak height, impedance peak width, peak area and / or half-width peak height.

[0236] Fig. 15C An embodiment of using impedance magnitude to determine the concentration of a target analyte is shown. Two impedance signal thresholds can be established. If the impedance signal exceeds a lower threshold but does not exceed an upper threshold, it is determined that the affinity portion passing through the nanopore or nanochannel is not bound to the target analyte. If the impedance signal exceeds both the lower threshold and the upper threshold, it is determined that the affinity portion passing through the nanopore or nanochannel is bound to the target analyte. The ratio of the bound affinity portion can be used to determine the analyte concentration in a biological sample.

[0237] A channel sensor (e.g., a flow cytometer or a channel sensor configured to measure analyte concentration) can be calibrated using particles of standardized size, surface charge, and / or material. Calibration can be done during manufacturing and improves the accuracy of determining cell size and type by accounting for electric field effects that may cause particle size errors. The channel sensor can additionally or alternatively be calibrated using various reagents with known pH and / or electrolyte concentrations, which can verify the conductivity of the pores.

[0238] In some embodiments, the channel sensor is calibrated during operation of the cartridge, for example before or after analysis of a biological sample. Calibration can be performed using reagents that are stored or mixed on the cartridge and delivered to the channel sensor using an electrowetting electrode.

[0239] In some embodiments, the box includes multiple cell analyzer sensors, such as two, three, four or more. Different cell analyzers can be redundant or dedicated. Using different cell analyzers, biological samples can be dynamically analyzed, such as by passing the biological sample through a first cell analyzer and then through a second cell analyzer, wherein the second cell analyzer is selected based on the results of the first cell analyzer. For example, if it is determined that the biological sample has a low cell count of a specific type of cell (e.g., white blood cells, red blood cells, or platelets), the biological sample can then be further processed by mixing with a specific reagent or by physically separating a specific cell type, and then analyzed using a cell analyzer with a different configuration (e.g., having a different pore size, functionalized with one or more molecules, or having a filter that removes certain cell types) to more accurately count specific types of cells.

[0240] Electrowetting electrode

[0241] The electrowetting electrodes in the box are configured to transport aqueous liquids (e.g., biological samples or processed biological samples) by electrowetting actuation (also known as digital microfluidics), divide the biological sample into one or more sub-samples, mix one or more reagents together, or mix one or more reagents with the biological sample or sub-sample to treat or dilute the biological sample or sub-sample. The electrowetting electrodes can, for example, transport the biological sample through the main chamber or channel of the box. Individual electrodes of the multiple electrowetting electrodes can, for example, be individually controlled by an electrowetting electrode driver in the device when engaged with the box. Using electrowetting electrodes to transport biological samples in the main chamber can increase the versatility of biological sample transport compared to channel-based fluid transport, which is linear and typically relies on capillary flow (allowing limited control) or moving parts, such as pumps, to transport samples. Electrowetting-based fluid transport can achieve precise movement of biological samples, reagent mixing, dilution, and sample splitting.

[0242] Electrowetting actuation is driven by applying a voltage to the fluid located above the activated electrode. The voltage changes the contact angle of the fluid by generating a charge on the fluid contact surface, which attracts the fluid to the activated electrode. In order to reduce the voltage required for electrowetting actuation, it is necessary to reduce the thickness of the insulating layer and / or the hydrophobic layer without causing dielectric breakdown of the material. Therefore, it is desirable to use a thin coating material with a high dielectric constant and high dielectric strength to reduce the voltage required for electrowetting actuation and generate the necessary charge on the fluid contact surface.

[0243] The electrowetting electrodes in the box can be arranged to form an array of spaced electrodes electrically connected to the device for operating the electrowetting electrodes. The device can use a switch to operate the electrowetting electrodes, which selectively operates the electrodes in the array to actuate liquids (e.g., reagents or biological samples). The electrowetting electrodes are separated from each other, and the insulating layer fills the space between the electrowetting electrodes. The space between the electrowetting electrodes can vary in the array according to the size of the electrowetting electrodes. For example, in some embodiments, the spacing between the electrodes is about 10 μm to about 150 μm (e.g., about 10 μm to about 20 μm, about 20 μm to about 50 μm, about 50 μm to about 100 μm or about 100 μm to about 150 μm). The size of the electrowetting electrode can also vary in the array according to the liquid volume to be occupied by the electrode. For example, the electrowetting electrode configured to actuate a liquid volume of 20 μL is about 0.632 mm by about 0.632 mm. In some embodiments, the electrowetting electrodes are approximately square or rectangular, although in some embodiments, the electrowetting electrodes may have jagged or protruding rounded edges with partial overlap between adjacent electrodes. Fig.16A An exemplary electrowetting electrode array is shown having an 8x16 matrix with traces electrically connecting each electrode to the device interface. Fig. 16B Magnified images of several electrowetting electrodes are shown, highlighting the jagged edges of the approximately square electrodes.

[0244] The electrowetting electrodes are coated with an insulating layer (i.e., a dielectric layer), which is preferably a high-κ material (i.e., having a dielectric constant of about 3.9 or higher). The insulating layer may also fill the space between the electrowetting electrodes. In some embodiments, the insulating layer has a dielectric constant of about 3.9 or higher (e.g., about 5 or higher, about 10 or higher, about 20 or higher, about 40 or higher, about 60 or higher, about 100 or higher, about 200 or higher, about 400 or higher, about 1000 or higher, or about 2000 or higher). In some embodiments, the insulating layer has a dielectric constant of about 3.9 to about 20,000 (e.g., about 3.9 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 40, about 40 to about 60, about 60 to about 100, about 100 to about 200, about 200 to about 400, about 400 to about 1000, about 1000 to about 2000, about 2000 to about 4000, about 4000 to about 6000, about 6000 to about 10,000, or about 10,000 to about 20,000). Exemplary materials for the insulating layer include or are hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide, or silicon nitride.

[0245] The insulating layer is preferably a thin layer deposited on the electrode surface. Minimize the thickness to reduce the required voltage without causing dielectric breakdown of the dielectric material. For example, the insulating layer can be applied to the electrode using atomic layer deposition, chemical vapor deposition, reactive ion beam deposition, sputtering deposition, evaporation, spray deposition, spin coating or sol-gel formation. In some embodiments, the insulating layer has a thickness of about 1nm to about 5μm. For example, in some embodiments, the thickness of the insulating layer is about 1nm to about 10nm, about 10nm to about 20nm, about 20nm to about 40nm, about 40nm to about 100nm, about 100nm to about 250nm, about 250nm to about 500nm, about 500nm to about 1μm or about 1μm to about 5μm. When the insulating layer includes a metal oxide, the insulating layer is generally thinner than when the insulating layer includes a polymer material. For example, in some embodiments, the insulating layer includes a metal oxide and has a thickness of about 1nm to about 100nm. In some embodiments, the insulating layer includes a polymer material and has a thickness of about 0.1μm to about 5μm. In some embodiments, the distance between the electrode and the fluid contact surface is from about 1 nm to about 25 μm. For example, in some embodiments, the distance between the electrode and the fluid contact surface is from about 1 nm to about 10 nm, from about 10 nm to about 20 nm, from about 20 nm to about 40 nm, from about 40 nm to about 100 nm, from about 100 nm to about 250 nm, from about 250 nm to about 500 nm, from about 500 nm to about 1 μm, from about 1 μm to about 5 μm, from about 5 μm to about 10 μm, or from about 10 μm to about 25 μm.

[0246] In some embodiments, the insulating layer has a surface (i.e., a fluid contact layer) in contact with a biological sample and / or reagent. The fluid contact surface is preferably a hydrophobic surface having a contact angle of 90° or more with water at 25°C. In some embodiments, the hydrophobic surface has a contact angle of about 90° to about 160° (e.g., about 90° to about 100°, about 100° to about 110°, about 110° to about 120°, about 120° to about 130°, about 130° to about 140°, about 140° to 150°, or 140° to 150°) with water at 25°C. In some embodiments, the insulating layer is or includes a hydrophobic material (i.e., a high-κ material that can be used as a hydrophobic insulating material). In order to further increase the hydrophobicity of the fluid contact surface of the insulating layer, the insulating layer may optionally include a nanostructured surface, which may be, for example, a woven pattern, a nanopillar pattern, or a nanograss pattern. The nanostructure pattern may be formed, for example, by etching into the insulating layer.

[0247] In some embodiments, the insulating layer is optionally coated with a hydrophobic material, which can be a fluid contact layer. In some embodiments, the hydrophobic layer is applied, for example, using thin film deposition or spin coating. Optionally, the fluid contact surface of the hydrophobic layer has a nanostructured surface, which can be, for example, a woven pattern, a nano-pillar pattern or a nano-grass pattern. Nanostructured patterns can be formed, for example, by etching into the hydrophobic layer, by wet etching or dry etching into the fluid contact layer (for example, the insulating layer or a separate hydrophobic layer). For example, photolithography or ion etching can be used to create a predefined pattern. Exemplary hydrophobic materials for hydrophobic layers include fluoropolymers, silanes, fluorinated silanes, polydimethylsiloxanes or polyparaxylene.

[0248] The fluid contact surface (which may be, for example, the surface of an insulating layer or a hydrophobic coating) may have a nanostructured surface (i.e., a nanostructured pattern on the fluid contact surface). The nanostructured surface may change the hydrophobicity of the surface, thereby reducing the voltage required to actuate the fluid on the electrowetting electrode. Exemplary nanostructured surfaces include, but are not limited to, a woven pattern, a nanopillar pattern, or a nanograss pattern. In some embodiments, the hydrophobic nanostructured patterned surface is patterned on the insulating surface so that no additional material coating is required. In other embodiments, a nanostructured hydrophobic surface is formed on top of one or more insulating layers to form a hydrophobic surface on the top insulating surface.

[0249] In some embodiments, a long chain molecule self-assembled on an insulating layer is used to form a hydrophobic layer. For example, the long chain molecule used for the hydrophobic layer can include a molecule with an alkane as a head group with or without a spacer portion and an end group specifically assembled on an insulating substrate, such as octadecyl hydroxamic acid, stearic acid, octadecylphosphonic acid, 16 hydrogen hexadecane hydrogen acid or octadecyl mercaptan. These materials form a hydrophobic layer on an insulating layer. For example, the thickness of the hydrophobic layer can be between about 2.5nm and about 30nm. This thin hydrophobic layer increases the hydrophobicity of the fluid contact surface without increasing the substantial thickness of the layer coating the electrowetting electrode.

[0250] The thin distance and high hydrophobicity of the surface in contact with the biological sample or reagent allow a lower voltage to be applied to the electrowetting electrode to allow electrowetting actuation. For example, in some embodiments, a plurality of electrowetting electrodes are configured to use a voltage of less than about 50 volts to transport the biological sample. In some embodiments, a plurality of electrowetting electrodes are configured to use a voltage of about 0.5 volts to about 50 volts (e.g., about 0.5 volts to about 1 volt, about 1 volt to about 5 volts, about 5 volts to about 5 volts to about 10 volts, about 10 volts to about 20 volts, about 20 volts to about 30 volts, about 30 volts to about 40 volts, or about 40 volts to about 50 volts) to transport the biological sample.

[0251] The plurality of electrowetting electrodes may include a plurality of coplanar electrodes that may be positioned along the bottom surface of the main chamber or channel of the box. Optionally, the box includes a ground electrode (which may be a continuous ground electrode or a plurality of separate ground electrodes) on the top surface of the main chamber or channel (i.e., on the opposite side of the coplanar electrode surface). As further described herein, the electrowetting electrodes may be coated with one or more layers, the top layer being a fluid contact layer that contacts the biological sample and / or reagent when they are transported within the box. Activation of each electrode modulates the hydrophobicity of the fluid contact layer above the electrode, thereby actuating the fluid (e.g., a biological sample or reagent) to a position above the activated electrode.

[0252] The electrowetting electrode may include any suitable conductive material, such as gold, silver, silver chloride, platinum, indium tin oxide (ITO), or conductive carbon. The conductive carbon may include conductive carbon ink, which may include carbon particles (e.g., graphite) mixed with other conductive particles, such as carbon silver ink.

[0253] Fig.17A An embodiment of a plurality of electrowetting electrodes including an insulating layer fluid contact surface is shown. The plurality of electrowetting electrodes include a first electrowetting electrode 1702, a second electrowetting electrode 1704, and a third electrowetting electrode 1706. On the surface opposite to the first electrowetting electrode 1702, the second electrowetting electrode 1704, and the third electrowetting electrode 1706, there is a ground electrode 1708 shared by the three electrowetting electrodes. An insulating layer 1710 covers the first electrowetting electrode 1702, the second electrowetting electrode 1704, and the third electrowetting electrode 1706 together with the space between the electrowetting electrodes. The ground electrode 1708 is also coated with an insulating layer 1712. Fluids such as biological samples and / or one or more reagents can be transported through the gap 1714 between the ground electrode 1708 and the first, second, and third electrowetting electrodes. The fluid contact surface 1716 of the insulating layer 1710 can be nanostructured to increase the hydrophobicity of the fluid contact surface. The fluid contacting surface 1718 of the insulating layer 1710 and / or the fluid contacting surface 1719 of the insulating layer 1712 may also be nanostructured to increase the hydrophobicity of the fluid contacting surface.

[0254] Fig. 17BAnother embodiment of a plurality of electrowetting electrodes is shown, which includes an insulating layer coating the electrowetting electrodes and a hydrophobic layer coating the insulating layer. The plurality of electrowetting electrodes include a first electrowetting electrode 1720, a second electrowetting electrode 1722, and a third electrowetting electrode 1724. On the surface opposite to the first electrowetting electrode 1720, the second electrowetting electrode 1722, and the third electrowetting electrode 1724, there is a grounding electrode 1726 common to the three electrowetting electrodes. The insulating layer 1728 covers the first electrowetting electrode 1720, the second electrowetting electrode 1722, and the third electrowetting electrode 1724 together with the space between the electrowetting electrodes. The grounding electrode 1726 is also coated with an insulating layer 1730. Coating the insulating layer 1728 is a hydrophobic layer 1732, and coating the insulating layer 1730 is a hydrophobic layer 1734. Fluid, such as a biological sample and / or one or more reagents, can be transported through gap 1736 between hydrophobic layers 1732 and 1734. Optionally, the fluid contacting surface of hydrophobic layer 1732 and / or the fluid contacting surface of hydrophobic layer 1734 is nanostructured to increase the hydrophobicity of the fluid contacting layer.

[0255] One or more of the electrowetting electrodes may include a single ground electrode opposite the electrowetting electrodes, instead of a common ground electrode. In some embodiments, a portion of the electrowetting electrodes include a common ground electrode, while a portion of the electrowetting electrodes include separate ground electrodes. Examples of these configurations are described in Fig. 17C (without a separate hydrophobic layer) and Fig.17D (with a separate hydrophobic layer).

[0256] In some embodiments, there is a method for transporting liquid, which includes placing an aqueous liquid on a first hydrophobic liquid contact surface above a deactivated first electrowetting electrode; activating the second electrowetting electrode by applying a voltage of less than about 50 volts to the second electrowetting electrode, thereby transporting the aqueous liquid from the first hydrophobic liquid contact surface to the second hydrophobic liquid contact surface above the second electrowetting electrode; wherein the first electrowetting electrode and the second electrowetting electrode are covered by an insulating layer and separated by the insulating layer.

[0257] The aqueous liquid can be a biological sample, such as blood, serum, saliva, sweat, tears, mucus, urine, or any other biological sample or derivative suspended in a fluid. In some embodiments, the liquid is a processed biological sample, which can be processed by mixing the liquid with one or more reagents. An electrowetting electrode can be used to mix the reagent with the biological sample. For example, in some embodiments, the method includes transporting the reagent to a liquid contact surface comprising the liquid, thereby mixing the reagent with the aqueous liquid.

[0258] The biological sample is transported using a voltage of about 50 volts or less through the electrowetting electrodes. For example, in some embodiments, the biological sample is transported using about 0.5 volts to about 50 volts (e.g., about 0.5 volts to about 1 volt, about 1 volt to about 2 volts, about 2 volts to about 3 volts, about 3 volts to about 4 volts, about 4 volts to about 5 volts, about 5 volts to about 10 volts, about 10 volts to about 20 volts, about 20 volts to about 30 volts, about 30 volts to about 40 volts, or about 40 volts to about 50 volts).

[0259] Methods for analyzing biological samples

[0260] The boxes and devices described herein can be used to analyze biological samples, such as determining blood cell counts or analyte concentrations. In order to analyze the sample, the biological sample is deposited in the box and multiple electrowetting electrodes (i.e., actuated by electrowetting) are used to transport the biological sample in the box. One or more sensors in the box are used to analyze the biological sample to generate analytical data, which is transmitted from the box (e.g., transmitted to a device engaged with the box). The biological sample can be mixed with one or more reagents in the box, such as to form a processed biological sample, which is analyzed using one or more sensors. The box can be discarded after a single use.

[0261] In some embodiments, the biological sample is received by the box by capillary action. The biological sample can fill a receiving chamber, which can determine the volume of the biological sample entering the box. After being analyzed by one or more sensors, the biological sample can be transported (e.g., by electrowetting actuation) to a waste container, where it can be stored until the box is disposed of.

[0262] The biological sample can be transported within the box (e.g., from a sample receiving port, to a mixing area, to one or more sensors, or to a waste container) by electrowetting actuation. The electrowetting electrodes can be coated with an insulating layer and / or a hydrophobic layer, either of which can include a hydrophobic fluid contact surface that can have a nanostructured pattern. In order to transport the biological sample, the first electrowetting electrode is activated by applying a voltage potential to the first electrowetting electrode to attract the biological sample to a position above the first electrode. Once the biological sample is in a position above the first electrode, the second electrowetting electrode is activated by applying a voltage to the second electrowetting electrode, and the first electrowetting electrode is deactivated, thereby transporting the biological sample to a position above the second electrowetting electrode. In some embodiments, the voltage is from about 0.5 volts to about 1000 volts (e.g., from about 0.5 volts to about 1 volt, from about 1 volt to about 5 volts, from about 5 volts to about 10 volts, from about 10 volts to about 20 volts, from about 20 volts to about 30 volts, from about 30 volts to about 50 volts, from about 50 volts to about 100 volts, from about 100 volts to about 250 volts, from about 250 volts to about 500 volts, or from about 500 volts to about 1000 volts). Preferably, the plurality of electrowetting electrodes are operated using about 50 volts or less, or more preferably about 20 volts or less.

[0263] In some embodiments, analyzing biological samples includes counting the number of cells in the biological samples. The analysis data transmitted by the box may include information related to the number of cells. In some embodiments, counting the number of cells includes distinguishing two or more different cell types, and the analysis data includes information related to the cell type and the number of cells. Different cell types may be, for example, leukocytes, erythrocytes or platelets. In some embodiments, different cell types may include eosinophils, basophils, neutrophils, monocytes and lymphocytes. Therefore, in some embodiments, analyzing biological samples includes counting leukocytes, counting erythrocytes, counting platelets, counting eosinophils, counting basophils, counting neutrophils, counting monocytes and / or counting lymphocytes. Analyzing biological samples may include applying an electric current to the biological sample and recording the multiplexed impedance of the electric current. Analysis data (i.e., multiplexed impedance) may be used to determine the number of cells or cell types in the biological sample. In some embodiments, analyzing biological samples includes at least one of the self-calibrating sensors to detect different cell sizes or different cell types.

[0264] In order to analyze biological samples using the cell analyzer sensor, the biological sample flows continuously through a channel including a microchannel or a micropore. When the biological sample flows through the micropore or microchannel, a multi-channel current is applied to the micropore or microchannel. The multiplexed electrical impedance in the micropore or microchannel is measured, and the analysis data related to the multiplexed electrical impedance is transmitted from the box. The electric current can be a mixed current including a DC component and multiple (e.g., 2, 3, 4, 5, 6, 7, 8 or more) AC components at different frequencies. Preferably, the multi-channel current includes at least three AC components. In some embodiments, the AC component includes a first AC current in a range of about 1 kHz to about 100 kHz (e.g., about 1 kHz to about 10 kHz, about 10 kHz to about 20 kHz, about 20 kHz to about 30 kHz, about 30 kHz to about 40 kHz, about 40 kHz to about 50 kHz, about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, or about 90 kHz to about 100 kHz), about 100 kHz to about 1 MHz (e.g., about 100 kHz to about 200 kHz, about 200 kHz to about 300 kHz, about 300 kHz to about 400 kHz, about 400 kHz to about 500 kHz), about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, or about 90 kHz to about 100 kHz). The present invention relates to a third alternating current of about 1 MHz or greater (e.g., about 1 MHz to about 10 MHz, about 10 MHz to about 20 MHz, about 20 MHz to about 30 MHz, about 30 MHz to about 40 MHz, about 40 MHz to about 50 MHz, about 50 MHz to about 60 MHz, about 60 MHz to about 70 MHz, about 70 MHz to about 80 MHz, about 80 MHz to about 90 MHz or about 90 MHz to about 100 MHz).In another embodiment, the multiplexed current comprises (1) a direct current or an alternating current of about 1 kHz to about 100 kHz (e.g., about 1 kHz to about 10 kHz, about 10 kHz to about 20 kHz, about 20 kHz to about 30 kHz, about 30 kHz to about 40 kHz, about 40 kHz to about 50 kHz, about 50 kHz to about 60 kHz, about 60 kHz to about 70 kHz, about 70 kHz to about 80 kHz, about 80 kHz to about 90 kHz, or about 90 kHz to about 100 kHz), (2) a direct current or an alternating current of about 50 kHz to about 2 (3) an alternating current of about 250 kHz to about 700 kHz (e.g., about 250 kHz to about 300 kHz, about 300 kHz to about 350 kHz, about 350 kHz to about 400 kHz, about 400 kHz to about 500 kHz), (4) an alternating current of about 50 kHz to about 70 kHz (e.g., about 70 kHz to about 90 kHz, about 90 kHz to about 110 kHz, about 110 kHz to about 130 kHz, about 130 kHz to about 150 kHz, about 150 kHz to about 200 kHz, or about 200 kHz to about 250 kHz), (5) an alternating current of about 250 kHz to about 700 kHz (e.g., about 250 kHz to about 300 kHz, about 300 kHz to about 350 kHz, about 350 kHz to about 400 kHz, about 400 kHz to about (4) an alternating current of about 700 kHz to about 5 MHz (e.g., about 700 kHz to about 800 kHz, about 800 kHz to about 900 kHz, or about 900 kHz to about 1 MHz, or about 1 MHz to about 2 MHz, about 2 MHz to about 3 MHz, about 3 MHz to about 4 MHz, or about 4 MHz to about 5 MHz); (5) an alternating current of about 5 MHz to about 20 MHz (e.g., about 5 MHz to about 10 MHz, about 10 In some embodiments, the plurality of AC components include an AC current of about 50 kHz to about 250 kHz, a second AC current of about 250 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, a fourth AC current of about 5 MHz to about 20 MHz, and a fifth AC current of about 20 MHz or more (e.g., about 20 MHz to about 30 MHz, about 30 MHz to about 40 MHz, about 40 MHz to about 50 MHz, about 50 MHz to about 60 MHz, about 60 MHz to about 70 MHz, about 70 MHz to about 80 MHz, about 80 MHz to about 90 MHz, about 90 MHz to about 100 MHz, 100 MHz to about 125 MHz, or about 125 MHz to about 150 MHz). In some embodiments, the plurality of AC components include a first AC current of about 50 kHz to about 250 kHz, a second AC current of about 250 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, a fourth AC current of about 5 MHz to about 20 MHz, and a fifth AC current of about 20 MHz to about 150 MHz. The number of cells and / or the number of two or more different cell types in the biological sample can be determined based on the measured multiplexed impedance. The impedance analyzed may include real and / or imaginary components.Alternatively, the analyzed impedance may include a magnitude component and / or a phase component.

[0265] In some embodiments, to analyze the sample, the biological sample is deposited into the box and the biological sample is transported within the box using a plurality of electrowetting electrodes (i.e., by electrowetting actuation). The biological sample is analyzed using one or more sensors within the box to generate analytical data, which is transmitted from the box (e.g., to a device engaged with the box). The biological sample can be mixed with one or more reagents within the box, for example, to form a processed biological sample, which is analyzed using one or more sensors. The box can be discarded after a single use.

[0266] In some embodiments, the biological sample is received by the box by capillary action. The biological sample can fill a receiving chamber, which can determine the volume of the biological sample entering the box. After being analyzed by one or more sensors, the biological sample can be transported (e.g., by electrowetting actuation) to a waste container, where it can be stored until the box is disposed of.

[0267] The biological sample can be transported within the cartridge (e.g., from a sample receiving port, to a mixing area, to one or more sensors, or to a waste container) by electrowetting actuation. To transport the biological sample, the first electrowetting electrode is activated by applying a voltage potential to the first electrowetting electrode to attract the biological sample to a position above the first electrode. Once the biological sample is in a position above the first electrode, the second electrowetting electrode is activated by applying a voltage to the second electrowetting electrode, and the first electrowetting electrode is deactivated, thereby transporting the biological sample to a position above the second electrowetting electrode.

[0268] In some embodiments, analyzing a biological sample includes determining the presence of an analyte (e.g., protein) in the biological sample or measuring the concentration of an analyte (e.g., protein). The biological sample can be statically positioned in a sensor (which can be, for example, an impedance sensor or an optical sensor) using, for example, electrowetting actuation (e.g., using an electrowetting electrode). The presence or concentration of an analyte can be determined in the following manner: the analyte in the biological sample is combined with an affinity portion that is bound to an electrode in the sensor, and the impedance change caused by the analyte being combined with the affinity portion is measured. Impedance changes can be calibrated, for example, for impedance caused by a reference solution that does not include a biological sample. The affinity portion can be, for example, an antibody, an antibody fragment, or an aptamer (e.g., a DNA aptamer, an RNA aptamer, or an XNA aptamer). The impedance detected by the sensor can be transmitted from the box, and impedance changes can be used to determine the presence or concentration of an analyte.

[0269] Although the embodiments of the present disclosure have been fully described with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications should be understood to be included within the scope of the examples of the present disclosure as defined by the appended claims.

[0270] Exemplary embodiments

[0271] The following embodiments are exemplary and should not be considered as limitations of the invention described herein.

[0272] Embodiment 1. A kit for analyzing a biological sample, comprising:

[0273] a sample receiving port configured to receive a biological sample;

[0274] a sensor configured to analyze the biological sample;

[0275] a cartridge space in fluid communication with the biological sample receiving port and the sensor;

[0276] a plurality of electrowetting electrodes configured to transport the biological sample within the cartridge space and combine the biological sample with one or more reagents; and

[0277] A device interface is configured to receive power from and communicate with a cartridge interface on the device, wherein the sensor and the plurality of electrowetting electrodes are in electrical communication with the device interface.

[0278] Embodiment 2. A box according to embodiment 1, wherein the box space is fluidly connected to one or more reagent containers.

[0279] Embodiment 3. A box according to embodiment 2, wherein the one or more reagent containers include reagents.

[0280] Embodiment 4. A box according to embodiment 2 or 3, wherein the box space includes a reagent mixing area.

[0281] Embodiment 5. A box according to any one of embodiments 1-4, wherein the box is a disposable box.

[0282] Embodiment 6. A box according to any one of embodiments 1-5, comprising: a waste container configured to receive the biological sample after being analyzed by the sensor.

[0283] Embodiment 7. The box according to any one of embodiments 1-6, wherein the biological sample enters the sample receiving port by capillary action.

[0284] Embodiment 8. A box according to any one of Embodiments 1-7, wherein the plurality of electrowetting electrodes comprises a plurality of coplanar electrodes.

[0285] Embodiment 9. A box according to embodiment 8, wherein the plurality of electrowetting electrodes further include a ground electrode on a surface opposite to the coplanar electrodes.

[0286] Embodiment 10. A box according to embodiment 9, wherein the ground electrode is common to two or more electrowetting electrodes.

[0287] Embodiment 11. A box according to embodiment 9, wherein at least one of the electrowetting electrodes is paired with a separate ground electrode.

[0288] Embodiment 12. A box according to any one of Embodiments 1-11, wherein the electrowetting electrode is coated with an insulating layer.

[0289] Embodiment 13. A box according to embodiment 12, wherein the insulating layer has a dielectric constant of about 3.9 or higher.

[0290] Embodiment 14. A box according to Embodiment 12 or 13, wherein the insulating layer comprises hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide or silicon nitride.

[0291] Embodiment 15. A box according to any one of Embodiments 12-14, wherein the insulating layer is coated on the electrode using atomic layer deposition, chemical vapor deposition, reactive ion beam deposition, sputtering deposition, evaporation, spray deposition, spin coating or sol-gel formation.

[0292] Embodiment 16. A box according to any one of Embodiments 12-15, wherein the insulating layer has a thickness of about 1 nm to about 5 μm.

[0293] Embodiment 17. A box according to any one of Embodiments 12-16, wherein the insulating layer is a fluid contact layer.

[0294] Embodiment 18. The cartridge of Embodiment 17, wherein the insulating layer comprises a nanostructured surface.

[0295] Embodiment 19. A box according to any one of Embodiments 1-18, wherein the electrowetting electrode is coated with a hydrophobic layer.

[0296] Embodiment 20. A box according to embodiment 19, wherein the hydrophobic layer is a fluid contact layer.

[0297] Embodiment 21. The box according to embodiment 20, wherein the hydrophobic layer comprises a nanostructured surface.

[0298] Embodiment 22. A box according to any one of Embodiments 19-21, wherein the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecylhydroxamic acid, stearic acid, octadecylphosphonic acid, 16-hydroxyhexadecylhydroaromatic acid, or octadecyl mercaptan.

[0299] Embodiment 23. A box according to any one of Embodiments 19-22, wherein the hydrophobic layer is coated on the insulating layer.

[0300] Embodiment 24. A box according to any one of Embodiments 1-23, wherein the electrowetting electrode comprises gold, silver, silver chloride, platinum, indium tin oxide or conductive carbon.

[0301] Embodiment 25. The box according to any of Embodiments 1-24, wherein the electrowetting electrode is separated from the fluid contacting surface by about 1 nm to about 25 μm.

[0302] Embodiment 26. A box according to any of Embodiments 1-25, wherein the plurality of electrowetting electrodes are configured to transport the biological sample using a voltage of less than about 50 volts.

[0303] Embodiment 27. A box according to any of embodiments 1-26, wherein the plurality of electrowetting electrodes are configured to transport the biological sample using a voltage of about 0.5V to about 50V.

[0304] Embodiment 28. A box according to any one of Embodiments 1-27, wherein the sensor is an impedance sensor.

[0305] Embodiment 29. The box according to any one of Embodiments 1-29, wherein the sensor is configured to detect a protein or measure the amount of the protein.

[0306] Embodiment 30. A box according to embodiment 28 or 29, wherein the sensor includes a first sensing electrode functionalized with an affinity portion, and a second sensing electrode paired with the first sensing electrode, wherein the sensor is configured to detect impedance changes when an analyte or protein binds to the affinity portion.

[0307] Embodiment 31. The box according to embodiment 30, wherein the affinity moiety is an antibody, an antibody fragment or an aptamer.

[0308] Embodiment 32. A box according to embodiment 30 or 31, wherein the first sensing electrode is an electrowetting electrode.

[0309] Embodiment 33. A box according to embodiment 30 or 31, wherein the sensor further includes a first electrowetting electrode and a second electrowetting electrode, wherein the first electrowetting electrode and the second electrowetting electrode are on opposite sides of the first sensing electrode or the second sensing electrode.

[0310] Embodiment 34. A box according to embodiment 33, wherein the first sensing electrode is an electrowetting electrode.

[0311] Embodiment 35. A box according to embodiment 33 or 34, wherein the plurality of electrowetting electrodes are configured to statically position at least a portion of the biological sample between the first sensing electrode and the second sensing electrode.

[0312] Embodiment 36. A box according to embodiment 34 or 35, wherein the first electrowetting electrode, the second electrowetting electrode and the first sensing electrode are electrically connected to a voltage switching circuit, and the voltage switching circuit is configured to selectively activate one of the first electrowetting electrode, the second electrowetting electrode or the first sensing electrode or deactivate any of them.

[0313] Embodiment 37. A box according to embodiment 36, wherein the voltage switching circuit is electrically connected to a switch configured to selectively select the impedance sensing circuit or the electrowetting electrode power supply circuit.

[0314] Embodiment 38. A box according to any one of Embodiments 28-37, wherein the impedance sensor includes a pH sensitive layer or an ion sensitive layer, which is configured to modulate impedance based on pH or ion concentration.

[0315] Embodiment 39. A box according to Embodiment 38, wherein the impedance sensor includes a metal oxide semiconductor capacitor (MOSCap) sensor.

[0316] Embodiment 40. A box according to Embodiment 39, wherein the MOSCap sensor is adjacent to the first sensing electrode and the second sensing electrode.

[0317] Embodiment 41. A box according to any one of Embodiments 1-40, wherein the box also includes one or more optical windows.

[0318] Embodiment 42. The cartridge of any one of Embodiments 1-41, wherein the sensor is a channel sensor comprising:

[0319] The first channel section,

[0320] The second channel section,

[0321] a hole or channel fluidly connecting the first channel segment and the second channel segment, and

[0322] An electrode pair is configured to apply current to the pore or channel and detect impedance within the pore or channel.

[0323] Embodiment 43. A box according to Embodiment 42, wherein the electrode pair is configured to directly contact the liquid flowing through the channel sensor.

[0324] Embodiment 44. A box according to Embodiment 42 or 43, wherein the channel sensor is a flow cytometer configured to count the number of cells in the biological sample.

[0325] Embodiment 45. A box according to embodiment 44, wherein the sensor is configured to distinguish between different types of cells.

[0326] Embodiment 46. A box according to embodiment 45, wherein the sensor is configured to distinguish between red blood cells, white blood cells and platelets.

[0327] Embodiment 47. A box according to embodiment 45 or 46, wherein the sensor is configured to count the number of red blood cells, white blood cells or platelets.

[0328] Embodiment 48. A box according to any of Embodiments 45-47, wherein the sensor is configured to distinguish between eosinophils, basophils, neutrophils, monocytes and lymphocytes.

[0329] Embodiment 49. A box according to any one of Embodiments 45-48, wherein the sensor is configured to count the number of eosinophils, the number of basophils, the number of neutrophils, the number of monocytes, or the number of lymphocytes.

[0330] Embodiment 50. The box according to any one of Embodiments 42-49, wherein the hole or channel is a microhole or microchannel.

[0331] Embodiment 51. A box according to any one of Embodiments 42-50, wherein the current is a multi-path current including multiple AC components at different frequencies.

[0332] Embodiment 52. A box according to any one of Embodiments 42-51, wherein the current is a multi-path current, and the multi-path current includes (1) a DC component or a low-frequency AC current, and (2) multiple AC components of different frequencies.

[0333] Embodiment 53. A box according to Embodiment 51 or 52, wherein the multiple AC components include at least three AC components.

[0334] Embodiment 54. A box according to embodiment 53, wherein the multiple AC components include a first AC current of about 10 kHz to about 100 kHz, a second AC current of about 100 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, and a fourth AC current greater than about 5 MHz.

[0335] Embodiment 55. A box according to any of Embodiments 51-54, wherein the multiple alternating currents include at least five different alternating current components.

[0336] Embodiment 56. A box according to embodiment 55, wherein the multiple AC components include a first AC current of about 50 kHz to about 250 kHz, a second AC current of about 250 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, a fourth AC current of about 5 MHz to about 20 MHz, and a fifth AC current of about 20 MHz to about 150 MHz.

[0337] Embodiment 57. A box according to any of Embodiments 42-56, wherein the electrode pair is configured to detect impedance at a sampling rate of about 100 kHz or higher.

[0338] Embodiment 58. A box according to any one of Embodiments 42-57, wherein the electrode pair is configured to detect real impedance components and imaginary impedance components within the hole or channel.

[0339] Embodiment 59. A box according to any one of Embodiments 42-57, wherein the electrode pair is configured to detect the amplitude impedance component and the phase impedance component within the hole or channel.

[0340] Embodiment 60. A cartridge according to Embodiment 42 or 43, wherein the channel sensor is configured to detect analyte concentration.

[0341] Embodiment 61. A box according to embodiment 60, wherein the analyte is a protein.

[0342] Embodiment 62. A box according to embodiment 60 or 61, wherein the box includes a reagent comprising an affinity portion, and wherein the box is configured to mix the reagent comprising the affinity portion with the biological sample or a subsample derived from the biological sample, and to transport the biological sample or the subsample to the channel sensor.

[0343] Embodiment 63. A box according to any one of Embodiments 60-62, wherein the hole or channel is a nanopore or nanochannel.

[0344] Embodiment 64. A box according to any one of Embodiments 1-63, wherein the box includes multiple sensors.

[0345] Embodiment 65. A box according to any one of Embodiments 1-64, wherein the biological sample is a blood sample.

[0346] Embodiment 66. A box according to any one of Embodiments 1-65, wherein the box space is a chamber or a channel.

[0347] Embodiment 67. A system for analyzing a biological sample, comprising:

[0348] A box according to any one of embodiments 1-66; and

[0349] A device includes a box interface configured to interface with the box, and the device is configured to power the box and operate the box.

[0350] Embodiment 68. A system according to Embodiment 67, wherein the device is configured to power and operate two or more different types of boxes.

[0351] Embodiment 69. A system according to embodiment 67 or 68, wherein the device also includes one or more processors and a non-transitory computer-readable storage medium storing one or more programs configured to be executed by the one or more processors, the one or more programs including instructions for (a) operating the multiple electrowetting electrodes in the box to transport the biological sample in the box, and (b) operating the sensor.

[0352] Embodiment 70. A system according to embodiment 69, wherein the one or more programs include instructions for operating multiple electrowetting electrodes in the box to continuously transport the biological sample through the sensor.

[0353] Embodiment 71. A system according to embodiment 70, wherein the one or more programs include instructions for operating multiple electrowetting electrodes in the box to statically position the biological sample within the sensor.

[0354] Embodiment 72. A system according to any one of embodiments 69-71, wherein the one or more programs include instructions for determining cell count or analyte concentration based on a multivariate pattern of impedance signals detected at one or more frequencies.

[0355] Embodiment 73. A system according to embodiment 72, wherein the multivariate mode includes one or more of impedance peak height, impedance peak width, impedance peak area, or impedance peak half-width peak height.

[0356] Embodiment 74. A system according to embodiment 72 or 73, wherein the multivariate mode includes a real impedance component and an imaginary impedance component.

[0357] Embodiment 75. A system according to embodiment 72 or 73, wherein the multivariate mode includes an amplitude impedance component and a phase impedance component.

[0358] Embodiment 76. A system according to any of Embodiments 67-75, wherein the one or more programs include instructions for calibrating a sensor.

[0359] Embodiment 77. A system according to any one of embodiments 67-76, wherein the device is a handheld device.

[0360] Embodiment 78. A method of analyzing a biological sample, comprising:

[0361] placing a biological sample into the cartridge;

[0362] transporting the biological sample within the cartridge using a plurality of electrowetting electrodes;

[0363] analyzing the biological sample using one or more sensors within the cartridge to generate analytical data; and

[0364] The analysis data is transmitted from the cartridge.

[0365] Embodiment 79. The method of embodiment 78, comprising mixing the biological sample with one or more reagents within the cartridge.

[0366] Embodiment 80. A method according to embodiment 78 or 79, wherein the biological sample is received by the box using capillary action.

[0367] Embodiment 81. The method of any one of Embodiments 78-80, further comprising transferring the biological sample to a waste container within the cartridge after analyzing the biological sample.

[0368] Embodiment 82. A method according to any one of Embodiments 78-81, wherein the box is disposed of after a single use.

[0369] Embodiment 83. A method according to any one of embodiments 78-82, wherein analyzing the biological sample includes counting the number of cells in the biological sample, and wherein the analysis data relates to the number of cells.

[0370] Embodiment 84. A method according to any one of embodiments 78-83, wherein analyzing the biological sample includes: distinguishing between two or more different cell types.

[0371] Embodiment 85. A method according to embodiment 84, wherein analyzing the biological sample includes distinguishing between red blood cells, white blood cells and platelets.

[0372] Embodiment 86. A method according to any one of embodiments 78-85, wherein analyzing the biological sample includes counting white blood cells, counting red blood cells, or counting platelets.

[0373] Embodiment 87. A method according to any one of embodiments 78-86, wherein analyzing the biological sample includes distinguishing between eosinophils, basophils, neutrophils, monocytes and lymphocytes.

[0374] Embodiment 88. A method according to any one of embodiments 78-87, wherein analyzing the biological sample includes counting the number of eosinophils, the number of basophils, the number of neutrophils, the number of monocytes, or the number of lymphocytes.

[0375] Embodiment 89. A method according to any one of embodiments 78-88, wherein analyzing the biological sample includes applying an electric current to the biological sample and recording a multiplexed impedance of the electric current.

[0376] Embodiment 90. A method according to Embodiment 89, wherein the current is a mixed current including multiple AC components of different frequencies.

[0377] Embodiment 91. A method according to Embodiment 89 or 90, wherein the current is a mixed current, which includes a DC component and multiple AC components at different frequencies.

[0378] Embodiment 92. A method according to any one of Embodiments 89-91, wherein the current is a mixed current, and the mixed current includes at least five AC components at different frequencies.

[0379] Embodiment 93. A method according to any one of Embodiments 78-92, comprising self-calibrating at least one of the sensors to detect different cell sizes.

[0380] Embodiment 94. A method according to any of Embodiments 78-93, wherein analyzing the biological sample includes continuously flowing the biological sample through at least one sensor during analysis.

[0381] Embodiment 95. A method according to any one of embodiments 78-94, wherein a voltage of about 50V or less is used to transport the biological sample within the box.

[0382] Embodiment 96. A method according to any one of embodiments 78-95, wherein a voltage of about 0.5V to about 50V is used to transport the biological sample within the box.

[0383] Embodiment 97. A method according to any one of embodiments 78-96, wherein analyzing the biological sample comprises determining the concentration of an analyte or protein within the biological sample.

[0384] Embodiment 98. A method according to Embodiment 97, wherein analyzing the biological sample includes statically placing the biological sample on at least one of the sensors.

[0385] Embodiment 99. A method according to Embodiment 98, wherein the biological sample is statically placed on the sensor using an electrowetting electrode.

[0386] Embodiment 100. The method of any one of embodiments 78-99, wherein determining the concentration of the analyte or protein in the biological sample comprises:

[0387] binding the analyte or protein to an affinity moiety that is bound to an electrode within one of the sensors, and

[0388] The change in impedance caused by the binding of the analyte or protein to the affinity moiety is measured.

[0389] Embodiment 101. A method according to embodiment 100, wherein the affinity portion is an antibody, an antibody fragment or an aptamer.

[0390] Embodiment 102. A system comprising (a) a cartridge configured to analyze a biological sample, the cartridge comprising:

[0391] (i) Sensors, including:

[0392] first channel section;

[0393] Second channel section;

[0394] a hole or channel fluidly connecting the first channel section and the second channel section;

[0395] an electrode pair configured to apply multiple currents or voltages to the pore or channel; and

[0396] an electrode pair configured to detect impedance at multiple frequencies within the pore or channel, wherein the electrode pair configured to apply multiple currents and the electrode pair configured to detect impedance are the same electrode pair or different electrode pairs; and

[0397] (ii) a device interface electrically connected to the sensor; and

[0398] (b) a device configured to interface with and operate the cartridge, the device comprising one or more processors and a non-transitory computer-readable storage medium storing one or more programs configured to be executed by the one or more processors, the one or more programs comprising instructions for determining a cell count or an analyte concentration based on the detected impedance, wherein the detected impedance comprises at least a first multivariate impedance pattern at a first frequency and a second multivariate impedance pattern at a second frequency.

[0399] Embodiment 103. A system according to embodiment 102, wherein the electrode pair configured to apply multi-path current and the electrode pair configured to detect impedance are the same electrode pair.

[0400] Embodiment 104. A system according to embodiment 102, wherein the electrode pair configured to apply multi-path current and the electrode pair configured to detect impedance are different electrode pairs.

[0401] Embodiment 105. A system according to any one of embodiments 102-104, wherein the first multivariate impedance mode and the second multivariate impedance mode each include a real component and an imaginary component of the impedance.

[0402] Embodiment 106. A system according to any one of embodiments 102-104, wherein the first multivariate impedance mode and the second multivariate impedance mode each include an amplitude component and a phase imaginary component of the impedance.

[0403] Embodiment 107. A system according to any one of embodiments 102-106, wherein the first multivariate impedance mode and the second multivariate impedance mode include one or more of the following: impedance peak height, impedance peak width, impedance peak area or impedance peak half-width peak height.

[0404] Embodiment 108. A system according to any of Embodiments 102-107, wherein the electrode pair is in direct contact with the interior of the channel.

[0405] Embodiment 109. A system according to any one of embodiments 102-108, wherein the sensor is configured to count the number of cells in the biological sample.

[0406] Embodiment 110. A system according to embodiment 109, wherein the sensor is configured to distinguish between different types of cells.

[0407] Embodiment 111. A system according to embodiment 110, wherein the sensor is configured to distinguish between red blood cells, white blood cells and platelets.

[0408] Embodiment 112. A system according to any one of embodiments 109-111, wherein the sensor is configured to count the number of red blood cells, the number of white blood cells, or the number of platelets.

[0409] Embodiment 113. A system according to any of embodiments 109-112, wherein the sensor is configured to distinguish between eosinophils, basophils, neutrophils, monocytes and lymphocytes.

[0410] Embodiment 114. A system according to any one of embodiments 109-113, wherein the sensor is configured to count the number of eosinophils, basophils, neutrophils, monocytes and lymphocytes.

[0411] Embodiment 115. A system according to any of Embodiments 109-114, wherein the pores or channels are micropores or microchannels.

[0412] Embodiment 116. A system according to any of embodiments 102-108, wherein the sensor is configured to detect an analyte concentration.

[0413] Embodiment 117. A system according to embodiment 116, wherein the analyte is a protein.

[0414] Embodiment 118. A system according to embodiment 116 or 117, wherein the pore or channel is a nanopore or nanochannel.

[0415] Embodiment 119. A system according to any of embodiments 102-118, wherein the electrode pair configured to detect impedance includes a first electrode within the first channel segment and a second electrode within the second channel segment.

[0416] Embodiment 120. A system according to any of embodiments 102-119, wherein the sensor includes the channel and wherein the electrode pair configured to detect impedance is located within the channel.

[0417] Embodiment 121. A system according to embodiment 120, wherein the electrode pair includes a first electrode near the first channel segment and a second electrode near the second channel segment.

[0418] Embodiment 121. A system according to embodiment 121, wherein the electrode pair includes a first electrode on the upper surface of the channel and a second electrode on the lower surface of the channel, and wherein the first electrode is located above the second electrode.

[0419] Embodiment 123. A system according to any of embodiments 102-122, wherein the sensor further includes an electrode pair configured to detect liquid flow within the sensor.

[0420] Embodiment 124. A system according to any of embodiments 102-123, wherein the sensor includes one or more isolated electrodes.

[0421] Embodiment 125. A system according to any one of Embodiments 102-124, wherein the sensor includes an inlet of the first channel segment near the electrowetting electrode.

[0422] Embodiment 126. A system according to any one of Embodiments 102-125, wherein the sensor includes an outlet of the second channel segment near the electrowetting electrode.

[0423] Embodiment 127. A system according to any one of embodiments 102-126, wherein the multi-path current includes multiple AC components at different frequencies.

[0424] Embodiment 128. A system according to any one of embodiments 102-127, wherein the multi-path current includes (1) a DC component or a low-frequency AC component, and (2) an AC component at a different frequency.

[0425] Embodiment 129. A system according to embodiment 127 or 128, wherein the multiple AC components include at least three AC components.

[0426] Embodiment 130. A system according to embodiment 129, wherein the multiple AC components include a first AC current of about 10 kHz to about 100 kHz, a second AC current of about 100 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, and a fourth AC current greater than about 5 MHz.

[0427] Embodiment 131. A system according to embodiment 127 or 128, wherein the multiple AC components include at least five AC components.

[0428] Embodiment 132. A system according to embodiment 131, wherein the multiple AC components include a first AC current of about 50 kHz to about 250 kHz, a second AC current of about 250 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, a fourth AC current of about 5 MHz to about 20 MHz, and a fifth AC current of about 20 MHz to about 150 MHz.

[0429] Embodiment 133. A system as described in any of embodiments 102-132, wherein the electrode pair configured to detect impedance is configured to detect impedance at a sampling rate of about 100 kHz or higher.

[0430] Embodiment 134. A system according to any of Embodiments 102-133, wherein the box includes a plurality of electrowetting electrodes, and the electrowetting electrodes are configured to transport one or more liquids within the box.

[0431] Embodiment 135. A system according to any of Embodiments 102-134, wherein the box is configured to mix the reagent with the biological sample or a subsample derived from the biological sample, and transport the biological sample or subsample to the sensor.

[0432] Embodiment 136. A method for determining a cell count in a biological sample, comprising:

[0433] transporting the biological sample through a sensor comprising a channel or a well;

[0434] applying multiple currents or voltages to the channel or pore;

[0435] detecting a multiplexed impedance within the channel or aperture, the multiplexed impedance comprising at least a first multivariate impedance mode at a first frequency and a second multivariate impedance mode at a second frequency; and

[0436] The cell count is determined based on the detected multiplexed impedance.

[0437] Embodiment 137. A method according to embodiment 136, wherein the first multivariate impedance mode and the second multivariate impedance mode each include a real component and an imaginary component.

[0438] Embodiment 138. A method according to embodiment 136, wherein the first multivariate impedance mode and the second multivariate impedance mode each include an amplitude component and a phase component.

[0439] Embodiment 139. A method according to any one of embodiments 136-138, wherein the multi-element impedance pattern includes one or more of impedance peak height, impedance peak width, impedance peak area, or impedance peak half-width peak height.

[0440] Embodiment 140. A method according to any one of embodiments 136-139, wherein the biological sample is in direct contact with a pair of electrodes for detecting multiplexed impedance.

[0441] Embodiment 141. A method according to any of Embodiments 136-140, comprising distinguishing two or more different types of cells.

[0442] Embodiment 142. A method according to any one of Embodiments 136-141, wherein determining the cell count includes determining a red blood cell count, a white blood cell count, or a platelet count in the biological sample.

[0443] Embodiment 143. A method according to any one of Embodiments 136-142, wherein determining the cell count includes determining an eosinophil count, a basophil count, a neutrophil count, a monocyte count, and a lymphocyte count in the biological sample.

[0444] Embodiment 144. A method according to any of Embodiments 136-143, comprising conveying two or more subsamples of the biological sample through the sensor, wherein the two or more subsamples are processed at different pH levels or different electrolyte concentrations.

[0445] Embodiment 145. A method according to any of Embodiments 136-144, comprising delivering a marker to the sensor.

[0446] Embodiment 146. A method according to embodiment 145, wherein the marker is an air bubble or a low conductivity solution.

[0447] Embodiment 147. The method of Embodiment 145 or 146, comprising detecting a marker.

[0448] Embodiment 148. A method according to any one of embodiments 136-147, wherein the marker triggers the start or end of recording of the detected multiplexed impedance.

[0449] Embodiment 149. A method according to any one of embodiments 136-148, comprising determining the flow rate of the biological sample.

[0450] Embodiment 150. A method according to Embodiment 149, comprising using the determined flow rate to determine the cell count.

[0451] Embodiment 151. A method according to any of Embodiments 136-150, comprising filtering the biological sample.

[0452] Embodiment 152. A method according to any one of embodiments 136-151, wherein the multi-path current includes multiple AC components at different frequencies.

[0453] Embodiment 153. A method according to any one of embodiments 136-152, wherein the multi-path current includes (1) a DC component or a low-frequency AC component, and (2) multiple AC components at different frequencies.

[0454] Embodiment 154. A method according to embodiment 152 or 153, wherein the multiple AC components include at least three AC components.

[0455] Embodiment 155. A method according to any one of Embodiments 152-154, wherein the multiple AC components include a first AC current of about 10 kHz to about 100 kHz, a second AC current of about 100 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, and a fourth AC current greater than about 5 MHz.

[0456] Embodiment 156. A system as described in any of Embodiments 152 or 153, wherein the plurality of AC components includes at least five AC components.

[0457] Embodiment 157. A system according to embodiment 156, wherein the multiple AC components include a first AC current of about 50 kHz to about 250 kHz, a second AC current of about 250 kHz to about 700 kHz, a third AC current of about 700 kHz to about 5 MHz, a fourth AC current of about 5 MHz to about 20 MHz, and a fifth AC current of about 20 MHz to about 150 MHz.

[0458] Embodiment 158. A method for detecting an analyte in a biological sample, comprising:

[0459] transporting the biological sample through a sensor comprising a channel or a well, the biological sample comprising an analyte bound to an affinity moiety;

[0460] applying a current or voltage to the channel or pore;

[0461] detecting impedance within the channel or pore; and

[0462] The analyte is detected based on the detected impedance.

[0463] Embodiment 159. A method according to embodiment 158, wherein the analyte is a protein or an electrolyte.

[0464] Embodiment 160. A method according to embodiment 158 ​​or 159, wherein the affinity moiety is a multivalent affinity moiety.

[0465] Embodiment 161. A method according to any one of embodiments 158-160, wherein the affinity portion is charged.

[0466] Embodiment 162. A method according to any one of Embodiments 158-151, wherein the affinity moiety comprises an aptamer, an antibody, or an antibody fragment.

[0467] Embodiment 163. A method according to any one of Embodiments 158-162, wherein the affinity portion comprises an aptamer that binds to avidin or an avidin derivative.

[0468] Embodiment 164. A method according to any one of Embodiments 158-163, comprising mixing the biological sample with a reagent comprising an affinity portion.

[0469] Embodiment 165. A method according to any one of embodiments 158-164, wherein detecting the analyte based on the detected impedance includes: distinguishing between the affinity portion bound to the analyte and the affinity portion that is not bound.

[0470] Embodiment 166. A method according to any of embodiments 158-165, comprising delivering a marker to the sensor.

[0471] Embodiment 167. A method according to embodiment 166, wherein the marker is an air bubble or a low conductivity solution.

[0472] Embodiment 168. The method according to embodiment 166 or 167, comprising detecting a marker.

[0473] Embodiment 169. A method according to any one of embodiments 166-168, wherein the marker triggers the start or end of recording of the detected multiplexed impedance.

[0474] Embodiment 170. A method according to any one of embodiments 166-169, comprising determining the flow rate of the biological sample.

[0475] Embodiment 171. A method according to embodiment 170, which includes using the determined flow rate to determine the concentration of the analyte.

[0476] Embodiment 172. An electrowetting electrode array comprising:

[0477] a plurality of coplanar electrowetting electrodes coated with and separated by an insulating layer;

[0478] Wherein the electrowetting array comprises a hydrophobic liquid contacting surface, and wherein the electrowetting array is configured to transport an aqueous liquid using a voltage of about 50 volts or less.

[0479] Embodiment 173. An electrowetting electrode array according to Embodiment 172, wherein the insulating layer includes a hydrophobic liquid contact surface.

[0480] Embodiment 174. An electrowetting electrode array according to Embodiment 173, wherein the insulating layer comprises a nanostructured surface.

[0481] Embodiment 175. An electrowetting electrode array according to Embodiment 172, wherein the insulating layer is coated with a hydrophobic layer comprising the hydrophobic liquid contact surface.

[0482] Embodiment 176. An electrowetting electrode array according to Embodiment 175, wherein the hydrophobic layer comprises a nanostructured surface.

[0483] Embodiment 177. An electrowetting electrode array according to embodiment 175 or 176, wherein the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecanehydroxamic acid, stearic acid, octadecanephosphonic acid, 16-hydroxyhexadecanehydrolamic acid or octadecanethiol.

[0484] Embodiment 178. An electrowetting electrode array according to any one of Embodiments 172-177, wherein the plurality of electrowetting electrodes are configured to transport the aqueous liquid using a voltage of about 0.5 volts to about 50 volts.

[0485] Embodiment 179. An electrowetting electrode array according to any one of Embodiments 172-178, wherein the insulating layer has a dielectric constant of about 3.9 or higher.

[0486] Embodiment 180. An electrowetting electrode array according to any one of Embodiments 172-179, wherein the insulating layer comprises hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide or silicon nitride.

[0487] Embodiment 181. An electrowetting electrode array according to any one of Embodiments 171-180, wherein the insulating layer is coated on the electrode using atomic layer deposition, chemical vapor deposition, reactive ion beam deposition, sputtering deposition, evaporation, spray deposition, spin coating or sol-gel formation.

[0488] Embodiment 182. An electrowetting electrode array according to any one of Embodiments 171-181, wherein the insulating layer has a thickness of about 1 nm to about 5 μm.

[0489] Embodiment 183. An electrowetting electrode array according to any one of Embodiments 171-182, wherein the electrowetting electrode comprises gold, silver, silver chloride, platinum, indium tin oxide or conductive carbon.

[0490] Embodiment 184. An electrowetting electrode array according to any one of Embodiments 171-183, wherein the electrowetting electrodes are separated from the hydrophobic liquid contact surface by about 1 nm to about 25 μm.

[0491] Embodiment 185. An electrowetting electrode array according to any one of Embodiments 171-184, wherein the plurality of electrowetting electrodes further comprise a ground electrode parallel to the coplanar electrowetting electrodes, wherein the hydrophobic liquid contact surface is between the ground electrode and the coplanar electrowetting electrodes.

[0492] Embodiment 186. An electrowetting electrode array according to Embodiment 185, wherein the ground electrode is common to two or more electrowetting electrodes.

[0493] Embodiment 187. An electrowetting electrode array according to embodiment 185, wherein at least one of the electrowetting electrodes is paired with a separate ground electrode.

[0494] Embodiment 188. An electrowetting electrode array according to any one of Embodiments 171-187, comprising an impedance sensor, wherein the impedance sensor comprises a sensing electrode and a functionalized liquid contact surface, wherein the functionalized liquid contact surface is functionalized with an affinity portion that specifically binds to a target analyte.

[0495] Embodiment 189. An electrowetting electrode according to embodiment 188, wherein the sensor further comprises a second sensing electrode paired with the first sensing electrode, wherein the sensor is configured to detect impedance changes when an analyte or protein binds to the affinity portion.

[0496] Embodiment 190. An electrowetting electrode array according to embodiment 188 or 189, wherein the target analyte is a protein.

[0497] Embodiment 191. An electrowetting electrode array according to any one of Embodiments 188-190, wherein the affinity portion is an antibody, an antibody fragment or an aptamer.

[0498] Embodiment 192. An electrowetting electrode array according to any one of Embodiments 188-191, wherein the impedance sensor also includes a metal oxide semiconductor capacitor (MOSCap) sensor, and the metal oxide semiconductor capacitor (MOSCap) sensor includes a pH-sensitive or ion-sensitive layer configured to modulate impedance based on pH or ion concentration.

[0499] Embodiment 193. An electrowetting electrode array according to any one of Embodiments 172-192, wherein the aqueous liquid comprises a biological sample.

[0500] Embodiment 194. An electrowetting electrode array according to embodiment 193, wherein the biological sample comprises a blood sample.

[0501] Embodiment 195. A kit for analyzing a biological sample, comprising:

[0502] a sample receiving port configured to receive a biological sample;

[0503] a sensor configured to analyze the biological sample;

[0504] A box space in fluid communication with a biological sample receiving port and a sensor, the box space comprising an electrowetting electrode array as described in any one of embodiments 172-194; and

[0505] A device interface is configured to receive power from and communicate with a cartridge interface on the device, wherein the sensor and the plurality of electrowetting electrodes are in electrical communication with the device interface.

[0506] Embodiment 196. A box according to Embodiment 195, wherein the box space is fluidly connected to one or more reagent containers, and wherein the electrowetting electrode array extends into the one or more reagent containers.

[0507] Embodiment 197. A box according to embodiment 195 or 196, wherein the electrowetting electrode array includes a reagent mixing area.

[0508] Embodiment 198. A box according to any one of Embodiments 195-197, wherein the box space is a chamber or a channel.

[0509] Embodiment 199. A system for analyzing a biological sample, comprising:

[0510] The box of any one of embodiments 195-198; and

[0511] A device comprising a cartridge interface configured to interface with the cartridge, the device being configured to operate the electrowetting electrode array.

[0512] Embodiment 200. A system according to embodiment 199, wherein the device is a handheld device.

[0513] Embodiment 201. A method of delivering a liquid, comprising:

[0514] placing an aqueous liquid on the first hydrophobic liquid contact surface above the inactivated first electrowetting electrode; and

[0515] activating the second electrowetting electrode by applying a voltage of about 50 volts or less to the second electrowetting electrode, thereby transporting aqueous liquid from the first hydrophobic liquid contact surface to a second hydrophobic liquid contact surface above the second electrowetting electrode;

[0516] The first electrowetting electrode and the second electrowetting electrode are covered by an insulating layer and separated by the insulating layer.

[0517] Embodiment 202. A method according to embodiment 201, wherein the aqueous liquid contains a biological sample.

[0518] Embodiment 203. A method according to Embodiment 201 or 202, comprising delivering a reagent to the second liquid contacting surface, thereby mixing the reagent with the aqueous liquid.

[0519] Embodiment 204. A method according to embodiment 203, wherein the reagent is delivered by activating a second electrowetting electrode.

[0520] Embodiment 205. A method according to any one of embodiments 201-204, wherein the biological sample is transported within the box using a voltage of about 0.5V to about 50V.

[0521] Embodiment 206. A method according to any one of Embodiments 201-205, wherein the first hydrophobic liquid contact surface or the second hydrophobic liquid contact surface is a nanostructured surface.

[0522] Embodiment 207. A method according to any one of Embodiments 201-206, wherein the insulating layer includes a first hydrophobic liquid contact surface and a second hydrophobic liquid contact surface.

[0523] Embodiment 208. A method according to any one of Embodiments 201-207, wherein the insulating layer is coated with a hydrophobic layer comprising a first hydrophobic liquid contact surface and a second hydrophobic liquid contact surface.

[0524] Embodiment 209. A method according to embodiment 208, wherein the hydrophobic layer comprises a fluoropolymer, polydimethylsiloxane, polyparaxylene, octadecylhydroxamic acid, stearic acid, octadecylphosphonic acid, 16-hydroxyhexadecylhydroaromatic acid, or octadecyl mercaptan.

[0525] Embodiment 210. A method according to any one of Embodiments 201-209, wherein the insulating layer has a dielectric constant of about 3.9 or higher.

[0526] Embodiment 211. A method according to any one of Embodiments 201-210, wherein the insulating layer comprises hafnium oxide, barium strontium titanate or strontium titanate, silicon dioxide or silicon nitride.

[0527] Embodiment 212. A method according to any one of Embodiments 201-211, wherein the insulating layer has a thickness of about 1 nm to about 5 μm.

[0528] Embodiment 213. A method according to any one of embodiments 201-212, wherein the first electrowetting electrode is separated from the first hydrophobic liquid contact surface by about 1 nm to about 25 μm, and the second electrowetting electrode is separated from the second hydrophobic liquid contact surface by about 1 nm to about 25 μm.

[0529] Embodiment 214. A metal oxide semiconductor capacitor (MOSCap) sensor comprising:

[0530] a first sensor segment comprising a first electrode and a first semiconductor layer;

[0531] a second sensor segment including a second electrode and a second semiconductor layer; and

[0532] a space between the first sensor segment and the second sensor segment configured to allow liquid flow;

[0533] The first sensor segment, the second sensor segment and the space are in a stacked structure.

[0534] Embodiment 215. A MOSCap sensor according to Embodiment 214, wherein the first semiconductor layer is directly coated on the first electrode, and the second semiconductor layer is directly coated on the second electrode.

[0535] Embodiment 216. A MOSCap sensor according to embodiment 214 or 215, wherein the first sensor segment includes a first insulating layer and the second sensor segment includes a second insulating layer.

[0536] Embodiment 217. A MOSCap sensor according to Embodiment 216, wherein the first insulating layer is directly coated on the first semiconductor layer, and the second insulating layer is directly coated on the second semiconductor layer.

[0537] Embodiment 218. A MOSCap sensor according to any one of embodiments 214-217, wherein the first sensor segment includes a first impedance modulation layer, and the second sensor segment includes a second impedance modulation layer, wherein the first impedance modulation layer and the second impedance modulation layer are pH-sensitive or ion-sensitive layers, which are configured to modulate impedance based on pH or ion concentration.

[0538] Embodiment 219. A MOSCap sensor according to embodiment 218, wherein the first sensor segment includes a first insulating layer, and the second sensor segment includes a second insulating layer; wherein the first impedance modulation layer is directly coated on the first insulating layer, and the second impedance modulation layer is directly coated on the second insulating layer.

[0539] Embodiment 220. A MOSCap sensor according to any one of Embodiments 214-219, wherein the first electrode is attached to a first substrate and the second electrode is attached to a second substrate.

[0540] Embodiment 221. A MOSCap sensor according to any one of Embodiments 214-220, wherein the first sensor segment is adjacent to a reference electrode.

[0541] Embodiment 222. A MOSCap sensor according to embodiment 221, wherein the first sensor segment is adjacent to the counter electrode.

[0542] Embodiment 223. A MOSCap sensor according to any one of Embodiments 214-222, wherein the MOSCap sensor is adjacent to an electrowetting electrode.

[0543] Embodiment 224. A MOSCap sensor according to Embodiment 223, wherein the electrowetting electrode is functionalized with an affinity moiety.

[0544] Embodiment 225. A MOSCap sensor according to Embodiment 224, wherein the affinity portion specifically binds hemoglobin. Example

[0545] The application can be better understood by reference to the following non-limiting examples provided as exemplary embodiments of the application. The following embodiments are given to more fully illustrate the embodiments, but should never be construed as limiting the broad scope of the application. Although some embodiments of the application have been shown and described herein, it is apparent that these embodiments are provided only by way of example. Without departing from the spirit and scope of the present invention, it will be appreciated by those skilled in the art that many variations, changes and replacements can be made. It should be understood that in practicing the apparatus and methods described herein, various alternatives to the embodiments described herein can be adopted.

[0546] Example 1: Cell size differentiation using flow cytometer channel sensors

[0547] The diameter of cells flowing through the flow cytometer can be determined using a low frequency current (e.g. <100 Hz), and the voltage signal can be calibrated to the signal modulation detected using polystyrene beads of known size. To calibrate the sensor, polystyrene beads of 5 μm, 10 μm, 20 μm, or 30 μm in diameter were suspended in phosphate buffered saline at pH 7.4 and passed through the flow cytometer channel sensor. A 30 kHz current (0.35 V pk ) passed through the sensor and the impedance was sampled at a rate of 460,000 samples per second. For each particle size, the voltage change over time is shown in Fig.18A In the graph, each peak reflects the passage of a single particle.

[0548] like Fig.18B As shown, the measured particle size is normalized to the true particle size to calibrate the measurement. Different particle sizes are linearly fit and the linear fit settings are used to calibrate future measurements using this flow cytometer channel sensor. To determine or calculate the size from the measured voltage signal, the voltage change relative to the reference signal (no particles) is converted to an impedance change. This change in impedance is converted to particle size using the theoretical relationship between channel size and impedance change, the formula is: where ΔZ is the impedance change relative to the baseline impedance Z without particles; d, L, D are the particle size, channel length, and channel diameter, respectively. is the correction factor.

[0549] Example 2: Determining Multiplexed Impedance Characteristics of Various Cell Types

[0550] The impedance characteristics of red blood cells, platelets, eosinophils, basophils, neutrophils, monocytes and lymphocytes were measured by isolating the various cell types and passing the cells through a flow cytometer channel sensor while measuring the impedance. The different cell types were separated using known techniques. The isolated cells were suspended in phosphate buffered saline at pH 7.4 and passed through a flow cytometer. The flow sensor passed a multi-channel current (0.35V pk ), the multi-channel current includes a low-frequency component (30kHz) for size detection and five other frequencies (107kHz, 570kHz, 1.55MHz, 16MHz and 40MHz), detecting the real and imaginary components of the impedance. The impedance signal is sampled at a rate of 460,000 samples per second, and a bandpass filter (8th order, 5Hz bandwidth) is used to filter and demodulate different impedance frequencies. Fig.19 Example real and imaginary impedances of a single eosinophil at all 5 frequencies are shown, with current flowing through the cell at -6.19 seconds. Although the data are not shown, similar plots were collected for other cell types. The impedance signals were normalized by dividing the amplitude of the 107kHz signal by all measurements (although the data could be normalized equally to any other frequency), and Fig. 20A (basophils), Fig. 20B (eosinophils), Fig. 20C (lymphocytes), Fig.20D (monocytes), Fig.20E (neutrophils), Fig.20F (platelets), Figure 20G The average normalized impedance of the real and imaginary impedance components at five different frequencies for different cell types are shown in Table 1 (red blood cells) and Table 1. In 20A-20G, points A–E on the radar chart correspond to frequencies of 107 kHz, 570 kHz, 1.55 MHz, 16 MHz, and 40 MHz, respectively.

[0551] Table 1: Normalized impedance amplitude characteristics

[0552]

[0553]

[0554] These impedance signatures are determined in a single environmental condition (e.g., pH, ion concentration of the buffer, etc.). By adjusting and modifying environmental variables (e.g., pH, ion concentration), environmental conditions can affect the surface and bulk properties of the cell, as well as the values ​​measured under those conditions. Therefore, additional impedance signatures can be collected under different buffer conditions to provide more unique characteristics of various cell types.

[0555] Example 3: Particle size classification in a flow sensor

[0556] A mixture of 5 μm and 10 μm polystyrene beads suspended in 1X PBS solution was pumped through the channel sensor at a constant flow rate of 2.5 μL / min. The channel sensor includes a microchannel (1 mm in length, 20 μm in width and height) with three electrodes spaced 12.5 μm apart along the length of the microchannel. As the beads flowed through the channel, an alternating current (0.5 Vpp, 100 kHz) was passed between the electrodes and the voltage change was measured. The voltage difference signal was normalized (the baseline was removed and the signal was demodulated from the alternating current so that the measured voltage was proportional to the impedance of the fixed alternating current signal). An exemplary normalized voltage signal over time is shown in Fig.21A , shows exemplary signals from 5 μm beads and 10 μm beads. When passing through the bead microchannel, the smaller pulse corresponds to the 5 μm beads and the larger pulse corresponds to the 10 μm beads. The histogram of the voltage difference signal is shown in Fig. 21B , and demonstrates two different classifications of bead sizes.

[0557] Example 4: Clustering of different cell types

[0558] As described in Example 2, the impedance characteristics of all single cells purified from whole blood of healthy males were previously collected. This data forms a labeled data set that allows the identification of single cell populations and clusters. Whole blood samples from healthy adults were diluted in 1X PBS to reduce the cell concentration and measured with a flow sensor. This unknown sample data is clustered into separate clusters across multiple parameters. These clusters are then further refined into categories or cell types using labeled data sets from previously collected data sets. A combination of unsupervised and supervised learning methods (i.e., semi-supervised learning techniques) is used to refine the classification. The trained model clusters the impedance characteristics into platelets, red blood cells (RBC), and white blood cells (WBC), such as Fig.22A As shown. Fig.22A In Figure 1, the classified cells are plotted and shown for 107kHz. At the same time, the same trained model allows further refinement and clustering of white blood cells based on impedance characteristics to distinguish between neutrophils, lymphocytes, and monocytes, as shown in Figure 1. Fig. 22B shown. Fig. 22B A diagram of the cell type at 1.55 MHz is shown. Fig. 22C An exemplary single signal versus time relationship is shown for the sensor in FIG. 1 , with each peak identified according to cell type (R for red blood cells, W for white blood cells, PT for platelets, and PS for 5 μm polystyrene beads added to whole blood to demonstrate discrimination).

[0559] After clustering the impedance signatures, counts for each cell type can be obtained. Table 2 shows the measured counts and the reference range for a healthy adult male.

[0560] Table 2

[0561]

[0562] Example 5: Measurement of hemoglobin in red blood cell lysate

[0563] The hemoglobin (Hb) content of red blood cell lysate was measured using a metal oxide semiconductor capacitor (MOSCap) sensor configured for hemoglobin detection. An Hb-specific aptamer with a thiol group at the 5' end of the aptamer was bound to an electrode. The electrode modified with the Hb-specific aptamer can be referred to as the "detection zone" of the sensor. Whole blood was mixed with a red blood cell lysis reagent, and the lysate was placed in the detection zone. The lysate was allowed to incubate in the detection zone, thereby allowing hemoglobin in the lysate to bind to the surface-bound Hb-specific aptamer. The detection zone was then washed three times with a wash buffer to remove the unbound portion of the lysate. A second Hb-specific aptamer conjugated with glucose oxidase (GOx) was moved to the detection zone, and the aptamer conjugated with GOx bound the hemoglobin bound to the surface-bound aptamer. The unbound GOx-conjugated aptamer was then removed by washing the detection zone. Glucose in the buffer was introduced into the detection zone, and GOx used the glucose to produce hydrogen peroxide and gluconic acid. The gluconic acid lowered the pH of the buffer, which was moved into the adjacent area of ​​the MOScap sensor where the sensor is sensitive to pH. The change in buffer pH is proportional to the concentration of hemoglobin in the sample. The hemoglobin concentration is determined based on calibration data using purified hemoglobin of known concentration.

Claims

1. A method for determining the cell count in a biological sample comprising: transporting the biological sample through a sensor comprising a channel or a well; applying multiple currents or voltages to the channel or pore; For a single cell in the biological sample, detecting a multiplexed impedance in the channel or well, the multiplexed impedance comprising at least a first multi-element impedance mode at a first frequency and a second multi-element impedance mode at a second frequency; as well as A cell count is determined based on the detected multiplexed impedance using a machine learning model executing on a processor that analyzes the detected multiplexed impedance, wherein the machine learning model uses a clustering algorithm.

2. The method according to claim 1, wherein: The first multivariate impedance mode and the second multivariate impedance mode each include a real component and an imaginary component.

3. The method according to claim 1, wherein: The first multivariate impedance mode and the second multivariate impedance mode each include an amplitude component and a phase component.

4. The method of claim 1, comprising distinguishing between two or more different types of cells.

5. The method according to claim 1, wherein: The multivariate impedance pattern includes one or more of impedance peak height, impedance peak width, impedance peak area, or impedance peak half-width peak height.

6. The method according to claim 1, wherein: The biological sample is in direct contact with a pair of electrodes that detect multiplexed impedance.

7. The method according to claim 1, wherein: Determining a cell count includes determining a red blood cell count, a white blood cell count, or a platelet count in a biological sample.

8. The method according to claim 1, wherein: Determining the cell count includes determining an eosinophil count, a basophil count, a neutrophil count, a monocyte count, and a lymphocyte count in the biological sample.

9. The method of claim 1, comprising passing two or more subsamples of the biological sample through the sensor, wherein the two or more subsamples are processed at different pH levels or different electrolyte concentrations.

10. The method according to claim 1, wherein: The multi-path current includes (1) a DC component or a low-frequency AC component, and (2) a plurality of AC components at different frequencies.

11. The method according to claim 1, wherein: The multi-path current includes three or more AC components at different frequencies.

12. The method according to claim 11, wherein: The three or more AC components include a first AC current of 10 kHz to 100 kHz, a second AC current of 100 kHz to 700 kHz, a third AC current of 700 kHz to 5 MHz, and a fourth AC current greater than 5 MHz.

13. The method according to claim 11, wherein: The three or more AC components include a first AC current of 50 kHz to 250 kHz, a second AC current of 250 kHz to 700 kHz, a third AC current of 700 kHz to 5 MHz, a fourth AC current of 5 MHz to 20 MHz, and a fifth AC current of 20 MHz to 150 MHz.

14. The method according to claim 11, wherein: The three or more AC components at different frequencies are used to distinguish different types of cells based on the unique impedance signature of each cell type.

15. The method according to claim 14, wherein: The unique impedance signature of each cell type is used to distinguish individual cells using a supervised machine learning model.

16. The method according to any one of claims 1 to 15, wherein: The sample is processed prior to analysis by merely diluting the sample and either without or with additional processing steps, including labeling of certain cell types or lysis of certain cells.

17. System, including (a) Sensors, including: first channel section; Second channel section; a hole or channel fluidly connecting the first channel segment and the second channel segment; an electrode pair configured to apply multiple currents or voltages to the pore or channel; as well as an electrode pair configured to detect impedance at multiple frequencies within the well or channel for a single cell in the biological sample, wherein the electrode pair configured to apply the multiplexed current and the electrode pair configured to detect impedance are the same electrode pair or different electrode pairs; as well as (b) one or more processors and a non-transitory computer-readable storage medium storing one or more programs configured to be executed by the one or more processors, the one or more programs comprising instructions for determining a cell count or analyte concentration based on the detected impedance using a machine learning model executed on the processor that analyzes the detected multiplexed impedance, wherein the machine learning model uses a clustering algorithm and wherein the detected impedance includes at least a first multivariate impedance pattern at a first frequency and a second multivariate impedance pattern at a second frequency.

18. The system of claim 17, wherein: The electrode pair configured to apply the multi-channel current and the electrode pair configured to detect the impedance are the same electrode pair.

19. The system of claim 17, wherein: The electrode pair configured to apply multi-channel current and the electrode pair configured to detect impedance are different electrode pairs.

20. The system of claim 17, wherein: The first multivariate impedance mode and the second multivariate impedance mode each include a real component and an imaginary component of the impedance.

21. The system of claim 17, wherein: The first multivariate impedance mode and the second multivariate impedance mode each include a magnitude component and a phase imaginary component of the impedance.

22. The system of claim 17, wherein: The first multi-element impedance mode and the second multi-element impedance mode include one or more of the following items: impedance peak height, impedance peak width, impedance peak area or impedance peak half-width peak height.

23. The system of claim 17, wherein: The multi-path current includes a plurality of alternating current components at different frequencies.

24. The system of claim 23, wherein: The plurality of AC components at different frequencies include three or more AC components.

25. The system of claim 17, wherein: The multi-path current includes (1) a DC component or a low-frequency AC component, and (2) multiple AC components at different frequencies.

26. The system of claim 23, wherein: The multi-path current includes three or more AC components, and the three or more AC components include a first AC current of 10kHz to 100kHz, a second AC current of 100kHz to 700kHz, a third AC current of 700kHz to 5MHz, and a fourth AC current greater than 5MHz.

27. The system of claim 23, wherein: The multi-channel current includes three or more AC components, and the three or more AC components include a first AC current of 50kHz to 250kHz, a second AC current of 250kHz to 700kHz, a third AC current of 700kHz to 5MHz, a fourth AC current of 5MHz to 20MHz, and a fifth AC current of 20MHz to 150MHz.

28. The system according to any one of claims 17 to 27, wherein: The sensor is configured to distinguish between different types of cells.

29. The system of claim 28, wherein: The sensor is configured to differentiate between red blood cells, white blood cells, and platelets.

30. The system of claim 28, wherein: The sensor is configured to differentiate between eosinophils, basophils, neutrophils, monocytes, and lymphocytes.

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