Systems, devices, and methods for processing biological samples

Abstract

Described are systems, methods, and devices to improve sample preparation approaches that can reliably generate ultra-long, high quality biological samples for nucleic acid sequencing. In an aspect, disclosed herein are methods for concentrating, purifying, preparing a library (or library prep), and extracting biomolecules from biological samples. Methods can include generating a fluidic flow of the sample through a channel, establishing an electric field along a length of the channel, concentrating the nucleic acids in a region comprising a constriction region of the channel, generating an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; isolating the region comprising the constriction region from other regions of the channel, and extracting the nucleic acids from the region comprising the constriction region.

Description

SYSTEMS, DEVICES, AND METHODS FOR PROCESSING BIOLOGICAL SAMPLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U. S. Provisional Application No. 63 / 602,787, filed November 27, 2023, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Sequencing of nucleic acids has emerged as an important technology in the field of genomics. Unlike short-read sequencing methods, long-read sequencing methods may have the capability to decipher larger nucleic acid fragments (e.g., > 18 kb) in their entirety. Such capability can allow scientists to bridge missing gaps between genomic regions and accurately analyze repetitive elements, structural variations, epigenetic modifications, and more. As sequencing methods become more cost-effective and accurate, their adoption may increase and unlock a deeper understanding of biological processes and their implications for human health.

[0003] However, while such methods associated with genomic sequencing and bioinformatics have rapidly evolved, unsolved technical problems remain in front-end preparation of biological samples for sequencing. As a result, sample preparation remains a costly, tedious, and timeconsuming process performed by skilled technicians and expensive instruments. Also, manual operations, e.g., handling and transferring biological samples, can result in costly loss, contamination, and degradation of biological samples. Sample preparation can be a particularly time-consuming process for long-read sequencing applications due to the inability to maintain ultra-long sample quality and length.

[0004] Recognized herein is an unmet need for systems, methods, and devices that can perform sample preparation of biological samples for sequencing.SUMMARY

[0005] Systems, methods, and devices herein can provide technical solutions in performing sample preparation of biological samples for sequencing. Sequencing can include short-read sequencing and long-read sequencing described herein. Disclosed herein are systems, methods, and devices to improve sample preparation approaches that can reliably generate ultra-long, high quality samples for nucleic acid sequencing. Systems, methods, and devices disclosed herein may also improve sample preparation for other biomolecules such as proteins.

[0006] In an aspect, disclosed herein is a method for extracting nucleic acids from a sample, comprising: (a) generating a fluidic flow of the sample through a channel, wherein the sample comprises the nucleic acids; (b) establishing an electric field along a length of the channel, wherein the electric field is configured to generate an electrophoretic flow that is opposite to the fluidicflow; (c) concentrating the nucleic acids in a region comprising a constriction region of the channel, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; (d) isolating the region comprising the constriction region from other regions of the channel, wherein substantially all of the concentrated nucleic acids remain in the region; and (e) extracting the nucleic acids from the region comprising the constriction region. In some embodiments, the isolating in (d) further comprises stopping the fluidic flow of the sample through the channel. In some embodiments, the stopping comprises closing valves to isolate the concentrated nucleic acids. In some embodiments, the valves comprise a pneumatic valve. In some embodiments, the valves are arranged to isolate the region comprising the constriction region. In some embodiments, the valves are arranged proximate to the region comprising constriction region. In some embodiments, the valves isolate the region comprising the constriction region and wherein the region contains a volume of about 10 microliters (pL). In some embodiments, the region comprising the constriction region consists essentially of a subsection of the channel comprising the restriction region. In some embodiments, the region comprising the constriction region: consists essentially of a subsection of the channel comprising the restriction region; and is isolatable from a remaining portion of the channel. In some embodiments, the region comprising the constriction region consists essentially of a subsection of the channel comprising the restriction region; and is isolatable from a remaining portion of the channel by a means comprising one or more valves. In some embodiments, the extracting in (e) further comprises using pressure to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some embodiments, the force is effected by applying pneumatic pressure to the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction region and into the collection region. In some embodiments, the extracting in (e) further comprises using an electrical force to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some embodiments, the electrical force is effected by applying an electrical field across or along the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction region and into the collection region. In some embodiments, the method further comprising coupling a collection device to the collection region, wherein the collection device is configured to extract the concentrated nucleic acids from the collection region. In some embodiments, the sample comprises the nucleic acids. In some embodiments, the sample comprises proteins. In some embodiments, the sample comprises a cell lysate sample. In some embodiments, the sample comprises a biological fluid. In some embodiments, the biological fluid comprises cellular nucleic acids. In some embodiments,the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample. In some embodiments, the sample comprises a buffer. In some embodiments, the buffer has an ionic concentration of about 0 millimolar (mM) to about 100 mM. In some embodiments, the buffer has an ionic concentration of greater than about 100 mM. In some embodiments, the buffer has a pH ranging from about 6.5 to about 8.5. In some embodiments, the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof. In some embodiments, the channel comprises a microchannel of a microfluidic device. In some embodiments, the microfluidic device comprises two or more instances of the microchannel, each comprising a constriction region configured to extract nucleic acids from a sample. In some embodiments, the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass. In some embodiments, the fluidic flow of the sample through the channel is at a volumetric flow rate of from about 0.001 microliters / minute (pL / min) to about 1000 pL / min. In some embodiments, the fluidic flow of the sample through the channel is at a flow speed of from about 0.001 millimeters / second (mm / s) to about 500 mm / s. In some embodiments, the electric field has a field strength of from about 0.001 millivolts / centimeter (mV / cm) to about 10 kilovolts / centimeter (kV / cm). In some embodiments, the electric field has a field strength; the fluidic flow has a volumetric flow rate; and the field strength and the volumetric flow rate establish a field strengthwolumetric flow rate ratio of about 4 volts / centimeter (V / cm): l pL / min. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate in an outer region of the region comprising the constriction region. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate at a top side portion, a bottom side portion, a left side portion, a right side portion, or any combination thereof of the region comprising the constriction region. In some embodiments, the region comprising the constriction region of the channel has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x wider than the channel. In some embodiments, the constriction region has a height ranging from about 0.001 nm to about 100 mm. In some embodiments, the constriction region has a height ranging from about 0.01 nm to about 10 mm. In some embodiments, the constriction region has a height ranging from about 0.1 nm to about 1 mm. In some embodiments, the constriction region has a height greater than 100 mm. In some embodiments, the constriction region has a height of about 225 micrometers (pm). In some embodiments, the constriction region comprises one or more corner regions each with a fillet radius ranging from about 0 pm to about 200 pm. In some embodiments, an entrance zone to the region comprising the constriction region has a shape selected from square,rectangular, circular, hyperbolic, or semi-circular. In some embodiments, the constriction region comprises one or more corner regions each with a fillet radius ranging from about 20 pm to about 50 pm. In some embodiments, an entrance zone to the region comprising the constriction region wherein the entrance zone has a shape selected from square, rectangular, circular, hyperbolic, and semi-circular. In some embodiments, the concentrated nucleic acids have an average length greater than about 300 kb. In some embodiments, the concentrated nucleic acids have an average length greater than about 500 kb. In some embodiments, the method further comprising analyzing a portion or substantially all of the concentrated nucleic acids. In some embodiments, the method further comprising sequencing a portion or substantially all of the concentrated nucleic acids. In some embodiments, the establishing in (b) further comprises: (a) applying a first voltage to an electrode at a first position; and (b) applying a second voltage to an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region, and wherein the electric field generated by the first and second voltages is substantially uniform along the length of the channel. In some embodiments, the first and second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow. In some embodiments, the first and second voltages are generated outside of the channel and electrically coupled to the channel. In some embodiments, the first and second voltages are generated proximate to the channel and electrically coupled to the channel. In some embodiments, the electrodes comprise microelectrodes integrated into the channel and electrically coupled to the channel. In some embodiments, the method further comprising heating the sample prior to or after the extracting in (e). In some embodiments, the method further comprising mixing the sample prior to or after the extracting in (e). In some embodiments, the method further comprising purifying the sample prior to or after the extracting in (e). In some embodiments, the method further comprising performing size selection on the sample prior to or after the extracting in (e)

[0007] In another aspect, disclosed herein is a device for extracting nucleic acids from a sample, comprising: a channel; a constriction region located along a length of the channel; a first valve coupled to the channel; a second valve coupled to the channel; and a collection region fluidically coupled to the constriction region. In some embodiments, the device further comprises: an inlet port fluidically coupled to the channel; an outlet port fluidically coupled to the channel; a fluid flow generator configured to generate a fluidic flow of the sample at a flow rate between the inlet port and the outlet port via the channel, wherein the sample comprises the nucleic acids; and an electric field generator configured to generate an electric field that causes an electrophoretic flow that is opposite to the fluidic flow, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force to concentrate the nucleic acids in a region comprising the constriction region thereby yielding concentrated nucleic acids. In someembodiments, the channel comprises a microchannel of a microfluidic device. In some embodiments, the microfluidic device comprises two or more instances of the microchannel each comprising a constriction region configured to extract nucleic acids from a sample. In some embodiments, the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass. In some embodiments, a length of the channel between the inlet port and the constriction region is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel between the inlet port and the constriction region is about 10 mm. In some embodiments, a length of the channel between an end of the constriction region to an opposite end of the constriction region is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel between an end of the constriction region to an opposite end of the constriction region is about 10 mm. In some embodiments, a length of the channel between an end of the constriction region to the outlet port is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel between an end of the constriction region to the outlet port is about 20 mm. In some embodiments, a width of the channel at regions other than the constriction region is from about 0.001 pm to about 10 mm. In some embodiments, a width of the channel at regions other than the constriction region is about 1 mm. In some embodiments, a width of the constriction region is from about 0.001 pm to about 100 mm. In some embodiments, a width of the constriction region is about 1 mm. In some embodiments, the constriction region has a height ranging from about 0.001 nm to about 100 mm. In some embodiments, the constriction region has a height ranging from about 0.01 nm to about 10 mm. In some embodiments, the constriction region has a height ranging from about 0.1 nm to about 1 mm. In some embodiments, the constriction region has a height greater than 100 mm. In some embodiments, the constriction region has a height of about 225 pm. In some embodiments, the constriction region of the channel has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 1 lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x wider than the channel. In some embodiments, the constriction region comprises one or more corners each with a fillet radius ranging from about 0 pm to about 200 pm. In some embodiments, the constriction region comprises one or more comers each with a fillet radius ranging from about 10 pm to about 100 pm. In some embodiments, the constriction region comprises one or more corners each with a fillet radius ranging from about 20 pm to about 50 pm. In some embodiments, an entrance zone to the region comprising the constriction region has a shape selected from square, rectangular, circular, hyperbolic, or semicircular. In some embodiments, the fluidic flow of the sample through the channel is at a volumetric flow rate of from about 0.001 pL / min to about 1000 pL / min. In some embodiments, the fluidic flow of the sample through the channel is at a flow speed of from about 1 pm / s to about500 mm / s. In some embodiments, the electric field has a field strength of from about 0.001 mV / cm to about 10 kV / cm. In some embodiments, the electric field has a field strength; the fluidic flow has a volumetric flow rate; and the field strength and volumetric flow rate establish a field strengthwolumetric flow rate ratio of about 4 V / cm: l pL / min. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate in an outer region of the region comprising the constriction region. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate at a top portion, a bottom portion, a left side portion, a right side portion, or any combination thereof of the region comprising the constriction. In some embodiments, the electric field generator generates a first voltage at an electrode at a first position and a second voltage at an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region. In some embodiments, the electric field generated by the first and second voltages is substantially uniform along the length of the channel. In some embodiments, the first and the second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow. In some embodiments, the first and second voltages are generated outside of the channel and electrically coupled to the channel. In some embodiments, the first and second voltages are generated proximate to the channel and electrically coupled to the channel. In some embodiments, the first and second voltages are generated at microelectrodes integrated into the channel and electrically coupled to the channel. In some embodiments, the device further comprises a third valve and a fourth valve, wherein the third valve and the fourth valve are configured to stop the fluidic flow of the sample through the channel. In some embodiments, the first valve and the second valve are configured to isolate the region comprising the constriction region from other regions of the channel thereby creating an isolated constriction region. In some embodiments, the first and second valves comprise a pneumatic valve. In some embodiments, the first and second valves are arranged to isolate the region comprising the constriction region. In some embodiments, the first and second valves are arranged proximate to the region comprising the constriction region. In some embodiments, the first and second valves isolate the region comprising the constriction region and wherein the region contains a volume of about 10 pL. In some embodiments, the device further comprising a third valve concentric with the constriction region and configured to extract the concentrated nucleic acids from a collection region. In some embodiments, the collection region is coupled to a collection device. In some embodiments, the collection device is configured to extract concentrated nucleic acids from the collection region. In some embodiments, the sample comprises the nucleic acids. In some embodiments, the sample comprises proteins. In some embodiments, the sample comprises a cell lysate sample. In some embodiments, the sample comprises a biological fluid. In some embodiments, the biological fluid comprises cellular nucleicacids. In some embodiments, the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample. In some embodiments, the sample comprises a buffer. In some embodiments, the buffer has an ionic concentration ranging from about 0 mM to about 100 mM. In some embodiments, the buffer has an ionic concentration of greater than about 100 mM. In some embodiments, the buffer has a pH ranging from about 6.5 to about 8.5. In some embodiments, the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof. In some embodiments, the device further comprising an element configured to heat the sample, wherein the element is integrated into the device or is coupled to the device. In some embodiments, the device further comprising an element configured to mix the sample, wherein the element is integrated into the device or is coupled to the device. In some embodiments, the device further comprising an element configured to purify the sample, wherein the element is integrated into the device or is coupled to the device.

[0008] In another aspect, disclosed herein is a computer program product for extracting nucleic acids from a sample, the computer program product comprising at least one non-transitory computer-readable medium having computer-readable program code portions embodied therein, the computer-readable program code portions comprising: (1) an executable portion configured generate a fluidic flow of the sample through a channel, wherein the sample comprises the nucleic acids; (2) an executable portion configured to establish an electric field along a length of the channel, wherein the electric field is configured to generate an electrophoretic flow that is opposite to the fluidic flow; (3) an executable portion configured to concentrate the nucleic acids in a region comprising a constriction region of the channel, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; (4) an executable portion configured to isolate the region comprising the constriction region from other regions of the channel, wherein substantially all of the concentrated nucleic acids remain in the region; and (5) an executable portion configured to extract the nucleic acids from the region comprising the constriction region.

[0009] Additional aspects and advantages of the present disclosure will become readily apparent from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the present disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0012] FIGs. 1A-1F illustrate principles of concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples using electrohydrodynamic forces, in accordance with some embodiments. FIG. 1 A illustrates a plan view of an example device for generating EH flows. FIG. IB illustrates a perspective view of a channel, an entrance zone, and a constriction region. FIG. 1C illustrates a plan view of an example of EH flow through a channel. FIG. ID illustrates an actual effect of using EH flows to concentrate or extract biomolecules. FIG. IE illustrates a perspective view that simulates an effect of using EH flows to concentrate or extract biomolecules. FIG. IF illustrates a migration or concentration of biomolecules in a channel.

[0013] FIGs. 2A-2C depict non-limiting examples of systems and devices for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, in accordance with some embodiments. FIG. 2A depicts an example system that can include equipment, instruments, or devices configured to implement methods disclosed herein. FIG. 2B depicts a device that can be integrated into a system for extracting biomolecules. FIG. 2C depicts an example device that can include the device in a stack of other devices such as heat sink, a controller, a thermal pad, a metal sheet, and a thermal cycler (e.g., a thermostat controller and a thermocouple).

[0014] FIG. 3 depicts a non-limiting example of a device (e.g., device 310) for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, in accordance with some embodiments.

[0015] FIGs. 4A-4D depict non-limiting examples of devices (e.g., device 410) and methods for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples,in accordance with some embodiments. FIG. 4A illustrates a plan view of a device. FIG. 4B illustrates a closer view of a constriction region. FIG. 4C illustrates an example workflow for performing any one or more methods. FIG. 4D illustrates a length, a width, or a height of a channel, an entrance zone, and a constriction region.

[0016] FIGs. 5A-5E depict non-limiting examples of a device (e.g., device 510), valves, and methods for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, in accordance with some embodiments. FIG. 5A illustrates a plan view of a device. FIG. 5B illustrates a closer view of a constriction region. FIG. 5C illustrates an example workflow for performing any one or more methods. FIG. 5D illustrates an example of a valve. FIG. 5E illustrates a length, a width, or a height of a channel, an entrance zone, and a constriction region.

[0017] FIGs. 6A-6B depict non-limiting examples of using or integrating devices disclosed herein with other devices configured to perform other operations, in accordance with some embodiments. FIG. 6A illustrates a stack of one or more devices with the cartridge positioned on the bottom side. Other devices can include a heat sink, a controller, a thermal pad, or a metal sheet. FIG. 6B further illustrates that other devices can include a thermostat controller and a thermocouple for measuring a temperate in the cartridge, e.g., inside the channel of the cartridge.

[0018] FIGs. 7A-7B depict non-limiting examples of using or integrating devices disclosed herein with other devices configured to perform other operations, in accordance with some embodiments. FIG. 7A illustrates a stack of one or more devices with the cartridge positioned on the top side. Other devices can include a heat sink, a controller, a thermal pad, or a metal sheet. FIG. 7B further illustrates that other devices can include a thermostat controller and a thermocouple for measuring a temperate in the cartridge, e.g., inside the channel of the cartridge.

[0019] FIG. 8 illustrates a non-limiting example of a computing device, in accordance with some embodiments.

[0020] FIG. 9 depicts a non-limiting example of a high-throughput device (HTD) for concentrating, preparing, and / or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, which can include certain inputs and outputs, in accordance with some embodiments. Preparing can include preparing a library (or library prep) for sequencing.

[0021] FIGs. 10A-10B depict a non-limiting example of a multiplexed device (MPD) for concentrating, preparing, and / or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, in accordance with some embodiments. Preparing can include preparing a library (or library prep) for sequencing. FIG. 10A illustrates a top view of the multiplexed device, which can include certain inputs and outputs. FIG. 10B illustrates different side views of the multiplexed device, which can be configured with levels (or layers) of different sized arrays.

[0022] FIGs. 11A-11F illustrate results of purifying, preparing, and / or extracting DNA using systems, methods, and devices herein, in accordance with some embodiments. Preparing can include preparing a library (or library prep) for sequencing. FIG. 11A illustrates that purification using systems, methods, and devices herein does not affect length distribution of DNA thereby demonstrating readiness for sequencing of DNA. FIG. 11B illustrates that purification using systems, methods, and devices herein does not affect length distribution of DNA thereby demonstrating readiness for sequencing of DNA. FIG. 11C illustrates that purification using systems, methods, and devices herein does not affect concentration of DNA thereby demonstrating readiness for sequencing of DNA. FIG. 11D illustrates purification performance as compared to other methods, e.g., solid-phase extraction using magnetic beads. FIG. HE illustrates purification performance as compared to other methods, e.g., solid-phase extraction using magnetic beads, using the A260 / 280 ratio. The A260 / 280 ratio is the ratio of ultraviolet (UV) absorbances of a samples at 260 nanometers (nm) and 280 nm. FIG. HF illustrates that purification using systems, methods, and devices herein does not affect sequencing of DNA.

[0023] FIGs. 12A-12E illustrate a non-limiting example workflow and results thereof for preparing DNA for ultra-long sequencing using systems, methods, and devices herein, in accordance with some embodiments. FIG. 12A illustrates the high-level workflow herein compared to other methods. FIGs. 12B-12E illustrate improved results of systems, methods, and devices herein compared to other methods.

[0024] FIGs. 13A-13D illustrate a non-limiting example workflow and results thereof for preparing DNA for DNA barcoding using systems, methods, and devices herein, in accordance with some embodiments. FIG. 13A illustrates the high-level workflow herein compared to other methods. FIGs. 13B-13D illustrate improved results of systems, methods, and devices herein compared to other methods.DETAILED DESCRIPTION

[0025] While various embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, or substitutions may occur without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0026] Disclosed herein are systems, methods, and devices that can improve sample preparation for generating ultra-long, high quality biological samples for nucleic acid sequencing. Systems, methods, and devices disclosed herein may also improve sample preparation for other biomolecules such as proteins. Compared to systems, methods, and devices herein, typicalmethods like magnetic bead purification and clean-up can require substantial manual labor, involve numerous error-prone steps, and generate considerable plastic waste. In contrast, by reducing steps and labor hours, systems, methods, and devices herein can reduce cost per sample by at least 30%, reduce preparation time per sample by at least 50%, and reduce manpower by at least 90%.Electrohydrodynamic flow for extracting biomolecules from biological samples

[0027] FIGs. 1A-1F illustrate principles of concentrating or extracting biological molecules (e.g., nucleic acids or proteins) from fluid samples (e.g., biological samples) using electrohydrodynamic (EH) flows, in accordance with some embodiments. See, e.g., Montes et. al, “Transverse migration and microfluidic concentration of DNA using Newtonian buffers,” Biomicrofluidics 13, 044104, 2019; Montes et. al, “Trapping DNA with a high throughput microfluidic device,” Electrophoresis 40, 437-446, 2019; Kopelevich et. al, “Mesoscopic models for electrohydrodynamic interactions of polyelectrolytes,” Journal of Fluid Mechanics 915, A95, 2021, each of which is incorporated by reference herein in its entirety.

[0028] In some cases, a sample can comprise a fluid sample. In some cases, the fluid sample can comprise one or more types of biological molecules. In some cases, the one or more types of biological molecules can comprise nucleic acids, deoxyribonucleic acids (DNA), ribonucleic acids (RNA), proteins, or any combination thereof. In some cases, a volume of the fluid sample can include a range of about 1 nanoliter (nL) to about 10 milliliters (mL). In some embodiments, the sample comprises the nucleic acids. In some embodiments, the sample comprises proteins. In some embodiments, the sample comprises a cell lysate sample. In some embodiments, the sample comprises a biological fluid. In some embodiments, the biological fluid comprises cellular nucleic acids. In some embodiments, the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample. In some embodiments, the sample comprises a buffer. In some embodiments, the buffer has an ionic concentration of about 0 mM to about 100 mM. In some embodiments, the buffer has an ionic concentration of greater than about 100 mM. In some embodiments, the buffer has a pH ranging from about 6.5 to about 8.5. In some embodiments, the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof.

[0029] FIG. 1A illustrates a plan view of an example device for generating EH flows. The device of FIG. 1A can include an input port 110, a channel 130, an entrance zone 135 of the channel 130, a constriction region 140 of the channel 130, and an output 120. The input port 110 can be configured to introduce the sample to the device or otherwise flow the sample into the device. The channel 130 can be configured to contain, seal, guide, or flow the sample from the input port 110to the output port 120. In some, cases, the channel 130 can include regions such as the entrance zone 135 or the constriction region 140. In some cases, the channel 130 may guide or flow the sample from the input port 110 to the output port 120 via the entrance zone 135 and the constriction region 140. FIG. IB illustrates a perspective view of the channel 130, the entrance zone 135, and the constriction region 140.

[0030] In some cases, an EH flow can include at least two flows. A first flow can include a convective flow. The convective flow can include a fluidic flow of the sample at a volumetric flow rate (e.g., pL / min through the channel 130) or a flow speed or velocity (e.g., mm / s along a centerline of the channel 130). In some cases, the convective flow may be generated by an internal fluid flow generator integrated into the device such as one or more micropumps configured to generate the fluidic flow of the sample through the device. In some cases, the convective flow may be generated by an external fluid flow generator such a one or more mechanical or electrical pumps configured to generate the fluidic flow of the sample through the device. In some cases, the convective flow may be generated by one or more combinations of internal and external fluid flow generators.

[0031] In some cases, the EH flow can include at least two flows. A second flow can include an electrophoretic flow, which can be generated by an electric field. The electrophoretic flow can include a flow or migration of a biological sample, which can include biomolecules (e.g., nucleic acids or proteins). In some cases, the electrophoretic flow of the biomolecules can be different than the convective flow. For example, as described herein elsewhere, the electrophoretic flow of the biomolecules can be different in the channel 130, the entrance zone 135, or the constriction region 140 than the convective flow. In some cases, the electrophoretic flow of the biomolecules may be similar or the same as the convective flow. For example, as described herein elsewhere, the electrophoretic flow of the biomolecules can be similar or the same in the channel 130, the entrance zone 135, or the constriction region 140 as the convective flow. In some cases, the electrophoretic flow of the biomolecules may be opposite to the convective flow in the channel 130, the entrance zone 135, or the constriction region 140. In some cases, the electrophoretic flow may be generated by an internal electric field generator integrated into the device such as one or more microelectrodes configured to generate the electric field. In some cases, the electrophoretic flow may be generated by an external electric field generator such as one or more electrical signal generators or signal amplifiers configured to generate the electric field. In some cases, the electrophoretic flow of the biomolecules established by the electric field may be generated by one or more combinations of internal and external electric field generators.

[0032] In some cases, the concentrating or extracting of biomolecules (e.g., nucleic acids or proteins) from biological samples may be improved by optimizing the electric field or the fluidicflow that is used to generate the EH flow. In some cases, the electric field may have a field strength (e.g., expressed in mV / cm or kV / cm). In some embodiments, the electric field has a field strength of from about 0.001 mV / cm to about 10 kV / cm. In some cases, the fluidic flow through the channel 130, the entrance zone 135, or the constriction region 140 may comprise a volumetric flow rate (e.g., expressed in pL / min) or a flow speed (e.g., expressed in mm / s). In some embodiments, the fluidic flow of the sample through the channel 130 is at a volumetric flow rate of from about 0.001 pL / min to about 1000 pL / min. In some embodiments, the fluidic flow of the sample through the channel 130 is at a flow speed of from about 0.001 mm / s to about 500 mm / s.

[0033] In some cases, the concentrating or extracting of biomolecules (e.g., nucleic acids or proteins) from biological samples may be improved by optimizing a ratio of the electric field to the fluidic flow that is used to generate the EH flow. In some cases, the ratio ranges from about (0.001 mV / cm: 1000 pL / min) to about (10 kV / cm:0.001 pL / min). In some embodiments, (a) the electric field has a field strength; (b) the fluidic flow has a volumetric flow rate; and (c) the field strength and the volumetric flow rate establish a field strength: volumetric flow rate ratio of about 4 V / cm: l pL / min. In some cases, different factors can be optimized, e.g., channel geometry, electric field strength, flow rates, and the like, to selectively extract nucleic acids from biological samples. For example, extraction of DNA can scale with DNA charge so longer DNA fragments can be extracted more quickly than shorter DNA fragments thereby allowing for size selection of DNA fragments. In some cases, devices herein can include two or more chambers thereby allowing DNA fragments to be collected in a first set of chambers and RNA fragments to be collected in a second set of chambers. In some cases, chambers can include channels of devices herein for collecting the DNA fragments and RNA fragments. In some cases, chambers can include portions separate from channels of devices herein for collecting the DNA fragments and RNA fragments.

[0034] FIG. 1C illustrates a plan view of an example of EH flow through the channel 130, which can comprise the entrance zone 135 or the constriction region 140. For example, biomolecules (e.g., nucleic acids or proteins) can enter the channel 130 due to a convective flow comprising a convective flow profile across or along the channel 130, the entrance zone 135, or the constriction region 140. An electric field applied along a centerline of the channel 130 can generate an electrophoretic flow having an electrophoretic flow profile speed or velocity across or along the channel 130. In some cases, the electrophoretic flow may be opposite from or opposed to the convective flow. In some cases, the electrophoretic flow speed or velocity can be at most about 20%, 10%, 5%, 1%, or less of the mean convective flow speed or velocity.

[0035] In some cases, the EH flow generates one or more forces, which can cause the biomolecules (e.g., nucleic acids or proteins) of the biological sample to migrate toward thebounding walls of the channel 130, the entrance zone 135, or the constriction region 140. In some cases, the migration can occur due to the electrophoretic flow of the biomolecules, the convective flow of the biomolecules, or a combination thereof. In some cases, the biomolecules can migrate toward a region of the centerline of the channel 130, the entrance zone 135, or the constriction region 140, or a combination thereof. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate in an outer region of the region comprising the constriction region 140. In some embodiments, the electrohydrodynamic force causes the nucleic acids to substantially concentrate at a top portion, a bottom portion, a left side portion, a right side portion, or any combination thereof of the region comprising the constriction region 140. For example, the biomolecules may migrate toward a centerline region of the channel 130, the entrance zone 135, or the constriction region 140 when the biomolecules are proximate to the bounding walls thereof and the electrophoretic flow speed or velocity exceeds the convective flow speed or velocity. In some cases, the migrating toward a centerline region of the channel 130, the entrance zone 135, or the constriction region 140 causes the biomolecules to concentrate or accumulate in a thin layer proximate to the bounding walls thereof.

[0036] FIG. ID is an example plan view that illustrates the actual effect of using EH flows to concentrate or extract biomolecules (e.g., nucleic acids or proteins) from biological samples near the channel 130, the entrance zone 135, or the constriction region 140. For example, devices and methods disclosed herein utilized EH flows to separate T4 DNA (image left) from microspheres (image right) in a sample. FIG. IE is a perspective view that simulates the effect of using EH flows to concentrate or extract biomolecules (e.g., nucleic acids or proteins) from biological samples near the channel 130, the entrance zone 135, or the constriction region 140 at a point in time, e.g., after 5 minutes of EH flow. As illustrated in FIG. IE, the biomolecules can migrate and concentrate toward a centerline region of the bounding walls of the channel 130, the entrance zone 135, or the constriction region 140. FIG. IF is a perspective view that simulates the effect of using EH flows to concentrate or extract biomolecules (e.g., nucleic acids or proteins) from biological samples near the channel 130, the entrance zone 135, or the constriction region 140 over a period of time, e.g., at 0, 3, and 5 minutes. As illustrated in FIG. IE, the biomolecules can migrate and concentrate toward a centerline region of the bounding walls of the channel 130, the entrance zone 135, or the constriction region 140.Methods and devices for extracting biomolecules from biological samplesDescription of methods and devices

[0037] In an aspect, disclosed herein is a method for extracting nucleic acids from a sample, comprising: (a) generating a fluidic flow of the sample through a channel, wherein the samplecomprises the nucleic acids; (b) establishing an electric field along a length of the channel, wherein the electric field is configured to generate an electrophoretic flow that is opposite to the fluidic flow; (c) concentrating the nucleic acids in a region comprising a constriction region of the channel, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; (d) isolating the region comprising the constriction region from other regions of the channel, wherein substantially all of the concentrated nucleic acids remain in the region; and (e) extracting the nucleic acids from the region comprising the constriction region.

[0038] In some embodiments, the isolating in (d) further comprises stopping the fluidic flow of the sample through the channel. For example, devices described herein elsewhere (e.g., device 310, 410, or 510) can use one or more valve actuators, one or more valves, and one or more valve actuator vias to stop or start the fluidic flow of the sample through the channel. Generally, the valve actuators can include a means for operating the valves. For example, the valve actuators may use pneumatic actuation or switching, hydraulic actuation or switching, or electrical actuation or switching to operate the valves (e.g., open or close the valves). Pneumatic actuation can include generating pneumatic pressure (e.g., air pressure) to operate the valves. In some embodiments, the valves comprise a pneumatic valve. Hydraulic actuation can include generating hydraulic pressure (e.g., fluid pressure) to operate the valves. Electrical actuation can include generating electrical pressure (e.g., electrostatic pressure) to operate the valves. The pressure (e.g., pneumatic pressure, hydraulic pressure, or electrical pressure) may be transmitted from the valve actuators to a flexible membrane of the valves through the valve actuator vias, which the flexible membrane is described herein elsewhere.

[0039] In some embodiments, the stopping comprises closing valves to isolate the concentrated nucleic acids. In some embodiments, the valves are arranged to isolate the region comprising the constriction region. In some embodiments, the valves are arranged proximate to the region comprising constriction region. For example, devices described herein elsewhere (e.g., device 310, 410, or 510) can use one or more valve actuators, one or more valves, and one or more valve actuator vias to isolate the concentrated nucleic acids. Generally, the valve actuators can include a means for operating the valves. For example, the valve actuators may use pneumatic actuation or switching, hydraulic actuation or switching, or electrical actuation or switching to operate the valves (e.g., open or close the valves). Pneumatic actuation can include generating pneumatic pressure (e.g., air pressure) to operate the valves. In some embodiments, the valves comprise a pneumatic valve. Hydraulic actuation can include generating hydraulic pressure (e.g., fluid pressure) to operate the valves. Electrical actuation can include generating electrical pressure (e.g., electrostatic pressure) to operate the valves. The pressure (e.g., pneumatic pressure, hydraulicpressure, or electrical pressure) may be transmitted from the valve actuators to a flexible membrane of the valves through the valve actuator vias, which the flexible membrane is described herein elsewhere. In some embodiments, the valves isolate the region comprising the constriction region, which contains a volume of about 10 pL. In some cases, the volume of the region can be a range of from about 0.1 pL to about 100 pL.

[0040] In some embodiments, the region comprising the constriction region (a) consists essentially of a subsection of the channel comprising the restriction region and (b) is isolatable from a remaining portion of the channel. In some embodiments, the region is isolatable from a remaining portion of the channel by a means comprising one or more valves. For example, devices described herein elsewhere (e.g., device 310, 410, or 510) can include an entrance zone and a constriction region along the length of the channel, which are configured to generate the EH flow. In some cases, the entrance zone can include the constriction region. The EH flow can extract or concentrate the biomolecules (e.g., nucleic acids or proteins) in the entrance zone, the constriction region, the channel, or a combination thereof. The valves can isolate the biomolecules in the entrance zone, the constriction region, the channel, or a combination thereof

[0041] In some cases, the biomolecules can comprise nucleic acids or proteins. The EH flow can extract or concentrate the biomolecules from a biological sample. In some cases, the concentrated nucleic acids have an average length ranging from about 1 kb to about 1000 kb or greater. In some embodiments, the concentrated nucleic acids have an average length greater than about 300 kb. In some embodiments, the concentrated nucleic acids have an average length greater than about 500 kb.

[0042] In some embodiments, the extracting in (e) further comprises using pressure to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel. For example, devices described herein elsewhere (e.g., device 310, 410, or 510) can include an extraction port or collection region. The extraction port or collection region may be fluidically or structurally coupled to the entrance zone, the constriction region, or the channel. In some cases, the extraction port or collection region may be fluidically or structurally coupled to a collection device. In some embodiments, the method further comprises coupling a collection device to the collection region, wherein the collection device is configured to extract the concentrated nucleic acids from the collection region. For example, the collection device can include a syringe to generate the pressure to force the concentrated biomolecules (e.g., nucleic acids or proteins) out of the collection region, the entrance zone, the constriction region, or the channel.

[0043] In some embodiments, the force is effected by applying pneumatic pressure to the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction regionand into the collection region. For example, devices described herein elsewhere (e.g., device 310, 410, or 510) can use one or more valve actuators, one or more valves, and one or more valve actuator vias to generate or apply a pressure to cause the concentrated nucleic acids to flow out of the entrance zone, the constriction region, or the channel and into the collection region. Generally, the valve actuators can include a means for operating the valves. For example, the valve actuators may use pneumatic actuation or switching, hydraulic actuation or switching, or electrical actuation or switching to operate the valves (e.g., open or close the valves). Pneumatic actuation can include generating pneumatic pressure (e.g., air pressure) to operate the valves. In some embodiments, the valves comprise a pneumatic valve. Hydraulic actuation can include generating hydraulic pressure (e.g., fluid pressure) to operate the valves. Electrical actuation can include generating electrical pressure (e.g., electrostatic pressure) to operate the valves. The pressure (e.g., pneumatic pressure, hydraulic pressure, or electrical pressure) may be transmitted from the valve actuators to a flexible membrane of the valves through the valve actuator vias, which the flexible membrane is described herein elsewhere.

[0044] In some embodiments, the extracting in (e) further comprises using an electrical force to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some embodiments, the electrical force is effected by applying an electrical field across or along the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction region and into the collection region. For example, an electric field may be generated to move the concentrated biomolecules out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some cases, the electric field can be the same electric field as the electric field for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the electric field can be another electric field configured to move the concentrated biomolecules out of the region comprising the constriction region and into a collection region that is proximate to the channel. For example, the electric field can be an AC or DC signal that is orthogonal or substantially orthogonal to the electric field for concentrating or extracting biomolecules. In some cases, the electric field can be an AC or DC signal that is parallel to or substantially parallel to the electric field for concentrating or extracting biomolecules. In some cases, the electric field generates an electrophoretic force to move the concentrated biomolecules out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some cases, the electric field generates a di electrophoretic force to move the concentrated biomolecules out of the region comprising the constriction region and into a collection region that is proximate to the channel. In some cases, the electric field generates a combination of electrophoretic force and di electrophoretic force tomove the concentrated biomolecules out of the region comprising the constriction region and into a collection region that is proximate to the channel.

[0045] In some embodiments, the method further comprises analyzing a portion or substantially all of the concentrated nucleic acids. For example, the concentrated biomolecules (e.g., nucleic acids or proteins) may be subject to further processing. Further processing can include analyzing the concentrated biomolecules. The analyzing can include, for example, quantitative or qualitative analysis. In some embodiments, the method further comprises sequencing a portion or substantially all of the concentrated nucleic acids. In some cases, sequencing can include shortread sequencing or long-read sequencing. For example, short-read sequencing can refer to short reads of less than about 500 base pairs (bp), 400 bp, 300 bp, 200 bp, 100 bp, and increments therein, or less. For example, long-read sequencing can refer to long reads of more than about 500 bp, 1000 bp, 10 kilobase pairs (kb), 100 kbp, 1000 kbp, and increments therein, or more. In some cases, systems, methods, and devices herein can be configured to prepare (e.g., concentrate, purify, prepare library, and extract) biomolecules for short-read sequencing or long-read sequencing.

[0046] In some embodiments, the establishing in (b) further comprises: (a) applying a first voltage to an electrode at a first position; and (b) applying a second voltage to an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region, and wherein the electric field generated by the first and second voltages is substantially uniform along the length of the channel. In some cases, the electric field generated by the first and second voltages can be substantially nonuniform along the length of the channel. In some cases, the electric field generated by the first and second voltages can be uniform or nonuniform along the length of the channel.

[0047] In some cases, devices described herein elsewhere (e.g., device 310, 410, or 510) can include an electric field generator configured to generate an electric field along the channel, which can be a uniform electric field, a nonuniform electric field, or a combination thereof. The electric field (e.g., uniform electric field) can generate an electrophoretic flow of the EH flow. In some embodiments, the first and second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow. In some embodiments, the first and second voltages are generated outside of the channel and electrically coupled to the channel. In some embodiments, the first and second voltages are generated proximate to the channel and electrically coupled to the channel. In some embodiments, the electrodes comprise microelectrodes integrated into the channel and electrically coupled to the channel.

[0048] In some embodiments, the method further comprises heating the sample prior to or after the extracting in (e). For example, devices described herein elsewhere (e.g., device 310, 410, or510) can include or be integrated with other devices configured to perform one or more other operations associated with concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. Other operations can include, for example, mixing, heating, cooling, purifying, or size selecting a biological sample. In some cases, the devices can be formed as a stack of devices, described herein elsewhere. For example, the stack can include devices such as a controller configured to control a temperature of a thermal pad for heating or cooling the sample. In some embodiments, the method further comprises mixing the sample prior to or after the extracting in (e). In some embodiments, the method further comprises purifying the sample prior to or after the extracting in (e).

[0049] In some embodiments, the method further comprises performing size selection on the sample prior to or after the extracting in (e). For example, the nucleic acids may be of different sizes or lengths. The EH flow may be configured to size or sort the nucleic acids based at least on their differences in sizes, lengths, or electrophoretic mobility. In some cases, the sizing or sorting may occur before the concentrating of the nucleic acids. In some cases, the sizing or sorting may occur after the concentrating of the nucleic acids. In some cases, the nucleic acids may have an average size or length ranging from about 1 kb to about 1000 kb or greater. In some cases, the sizing or sorting may size or sort the nucleic acids with a resolution of at most about 1000 kb, 100 kb, 10 kb, 1 kb, or less. For example, the sizing or sorting may size or sort the nucleic acids into groupings of nucleic acids with an average length of about 1000 kb, 100 kb, 10 kb, 1 kb, or less.Description of devices such as device 310

[0050] In another aspect, disclosed herein is a device for extracting nucleic acids from a sample, comprising: a channel; a constriction region located along a length of the channel; a first valve coupled to the channel; a second valve coupled to the channel; and a collection region fluidically coupled to the constriction region. In some embodiments, the isolating in further comprises stopping the fluidic flow of the sample through the channel.

[0051] In some embodiments, the device, further comprises: an inlet port fluidically coupled to the channel; an outlet port fluidically coupled to the channel; a fluid flow generator configured to generate a fluidic flow of the sample at a flow rate between the inlet port and the outlet port via the channel, wherein the sample comprises the nucleic acids; and an electric field generator configured to generate an electric field that causes an electrophoretic flow that is opposite to the fluidic flow, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force to concentrate the nucleic acids in a region comprising the constriction region thereby yielding concentrated nucleic acids.

[0052] In some cases, the terms device, cartridge, and cartridge assembly can be used interchangeably. In some cases, the cartridge assembly can include the device or cartridge. Insome cases, the cartridge assembly can include the device or cartridge and other devices configured together to concentrate or extract biological molecules (e.g., nucleic acids or proteins) from fluid samples (e.g., biological samples). In some cases, the terms electrohydrodynamic trapping (EHT) can be used interchangeably with purification.

[0053] FIG. 2B depicts an example cartridge assembly 300 that can be configured to implement any one or more methods disclosed herein. For example, the cartridge assembly 300 can include a cartridge holder 320 and a device, e.g., device 310, 410, or 510. The cartridge holder 320 may be configured to accept, retain, position, or align the device 310 to another device (e.g., equipment or instruments). In some cases, the cartridge holder 320 may be configured with a viewing window to view the device 310 from a position (e.g., above, below, or side) relative to the device 310. In some cases, the viewing can include optical viewing, infrared (IR) viewing, or ultraviolet (UV) viewing of biological samples in the device 310. In some cases, the cartridge holder 320 may be configured to reduce, suppress, or eliminate electromagnetic interference. In some cases, the device 310 may be configured to snap into the cartridge holder 320, plug and play into the cartridge holder 320, or fasten (e.g., screws) into the cartridge holder 320. In some cases, the cartridge holder 320 may be fabricated from a suitable material for the operation of device 310. For example, suitable materials can include plastics or metals that do not interfere with the operation of device 310.

[0054] In some cases, the device 310 may be electrically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include electrical connections associated with generating electric fields for EH flows, electrical connections associated with equipment or instruments to measure the concentrating or extracting of biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the device 310 may be mechanically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include mechanical connections associated with accepting, retaining, positioning, or aligning the device 310 to another device (e.g., equipment or instruments). In some cases, the device 310 may be fluidically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include fluidic connections associated with generating fluidic flows for EH flows to concentrate or extract biomolecules from biological samples. In some cases, the device 310 may be pneumatically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include pneumatic connections associated with actuating valves on the device 310 or generating fluidic flows for EH flows to concentrate or extract biomolecules from biological samples.

[0055] FIG. 3 depicts different embodiments of the device 310 that can be configured to implement any one or more methods disclosed herein. FIG. 3 illustrates a plan view of the device 310, which can have a nonlimiting footprint of 35 mm wide by 50 mm long. The device 310 caninclude an input port 310, an output port 320, a channel 330, an entrance zone 335 of the channel 330, a constriction region 340 of the channel 330, a valve actuator (not shown), a valve 355, a valve actuator via (not shown), an extraction port or collection region 380, or an extraction port via 385.

[0056] In some cases, the device 310 can include the input port 310 configured to introduce the sample to the device 310 or otherwise flow the sample into the device 310. The input port 310 may be fluidically or structurally coupled to the entrance zone 335, the constriction region 340, the channel 330, and the output port 320. The device 310 can include the output port 320 configured to remove the sample from the device 310 or otherwise flow the sample out of the device 310. The output port 320 may be fluidically or structurally coupled to the entrance zone 335, the constriction region 340, the channel 330, and the input port 310. The device 310 can include the channel 330 configured to contain, seal, guide, or flow the sample from the input port 310 to the output port 320. The channel 330 may be fluidically or structurally coupled to the input port 310, the entrance zone 335, the constriction region 340, and the output port 320. The device 310 can include the channel 330 configured to contain, seal, guide, or flow the sample from the input port 310 to the output port 320. In some, cases, the channel 330 can include regions such as the entrance zone 335 or the constriction region 340. In some cases, the channel 330 may guide or flow the sample from the input port 310 to the output port 320 via the entrance zone 335 and the constriction region 340.

[0057] In some cases, the device 310 can include the entrance zone 335 configured to flow the sample to the constriction region 340 with a flow profile. The entrance zone 335 may be fluidically or structurally coupled to the input port 310, the constriction region 340, the channel 330, and the output port 320. In some cases, the entrance zone 335 may be used interchangeably with the constriction region 340. In some cases, as illustrated in FIG. 1C, the combination of the entrance zone 335 and the constriction region 340 may be designed to generate a fluidic flow profile (e.g., convective flow) with a flow speed or velocity gradient across or along the channel 330, the constriction region 340, or the channel 330. For example, the entrance zone 335 may be designed with a width, length, and height that is different than the width, length, or height of the constriction region 340 or of the channel 330. FIG. 4D illustrates the widths, lengths, and heights. The difference in the width, length, or height may generate a flow profile (e.g., the convective flow) that operates in combination with the electrophoretic flow to concentrate or extract biomolecules (e.g., nucleic acids or proteins) from biological samples near the channel 330, the entrance zone 335, or the constriction region 340, as described herein elsewhere. For example, the entrance zone 335 and the constriction region 340 may be designed with a shape that helps to optimize the generating of the fluidic flow profile. In some embodiments, an entrance zone 335 to the regioncomprising the constriction region 340 has a shape selected from square, rectangular, circular, hyperbolic, or semi-circular.

[0058] In some cases, the width, length, or height of the entrance zone 335 may be designed based at least on generating a ratio of the width, length, or height of the entrance zone 335 to a width, length, or height of the channel 330. In some cases, the width, length, or height may be designed based at least on generating a ratio of the width, length, or height of the constriction region 335 to a width, length, or height of the channel 330. In some embodiments, the region comprising the constriction region 340 of the channel 330 has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, llx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x wider than the channel 330.

[0059] In some cases, the entrance zone 335 may be designed with one or more widths, lengths, or heights. In some cases, the width, length, or height of the entrance zone 335 may include the width length, or height of the constriction region 340. In some cases, the width of the entrance zone 335 is from about 0.001 pm to about 100 mm. In some cases, the width of entrance zone 335 is about 1 mm. In some cases, the length of the entrance zone 335 is from about 0.001 pm to about 100 mm. In some cases, the length of the entrance zone 335 is about 10 mm. In some cases, the entrance zone 335 has a height ranging from about 0.001 nm to about 100 mm. In some cases, the entrance zone 335 has a height ranging from about 0.01 nm to about 10 mm. In some cases, the entrance zone 335 has a height ranging from about 0.1 nm to about 1 mm. In some cases, the entrance zone 335 has a height greater than 100 mm. In some cases, the entrance zone 335 has a height of about 225 pm.

[0060] In some cases, the device 310 can include the constriction region 340. The constriction region 340 may be fluidically or structurally coupled to the input port 310, the entrance zone 335, the channel 330, and the output port 320. In some cases, the constriction region 340 may be designed with one or more widths, lengths, or heights. In some cases, the width, length, or height of the constriction region 340 may include the width length, or height of the entrance zone 335. In some embodiments, a width of the constriction region 340 is from about 0.001 pm to about 100 mm. In some embodiments, a width of the constriction region 340 is about 1 mm. In some embodiments, a length of the channel 330 between an end of the constriction region 340 to an opposite end of the constriction region 340 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 330 between an end of the constriction region 340 to an opposite end of the constriction region 340 is about 10 mm. In some embodiments, the constriction region 340 has a height ranging from about 0.001 nm to about 100 mm. In some embodiments, the constriction region 340 has a height ranging from about 0.01 nm to about 10 mm. In some embodiments, the constriction region 340 has a height ranging from about 0.1 nm to about 1 mm.In some embodiments, the constriction region 340 has a height greater than 100 mm. In some embodiments, the constriction region 340 has a height of about 225 gm.

[0061] In some cases, the device 310 can include the channel 330 having one or more regions other than the entrance zone 335 or the constriction region 340. The one or more other regions may be fluidically or structurally coupled to the input port 310, the entrance zone 335, the constriction region 340, the channel 330, and the output port 320. In some cases, the one or more other regions of the channel 330 (e.g., regions other than the entrance zone 335 or the constriction region 340) may be designed with one or more widths, lengths, or heights. In some embodiments, a width of the channel at regions other than the entrance zone 335 or the constriction region 340 is from about 0.001 pm to about 10 mm. In some embodiments, a width of the channel 330 at regions other than the entrance zone 335 or the constriction region 340 is about 1 mm. In some embodiments, a length of the channel 330 between the inlet port 310 and the constriction region 340 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 330 between the inlet port 310 and the constriction region 340 is about 10 mm. In some embodiments, a length of the channel 330 between an end of the constriction region 340 to the outlet port 320 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 330 between an end of the constriction region 340 to the outlet port 320 is about 20 mm. In some cases, the channel 330 at regions other than entrance zone 335 or the constriction region 340 has a height ranging from about 0.01 nm to about 10 mm. In some cases, the channel 330 at regions other than entrance zone 335 or the constriction region 340 has a height ranging from about 0.1 nm to about 1 mm. In some cases, the channel 330 at regions other than entrance zone 335 or the constriction region 340 has a height greater than 100 mm. In some cases, the channel 330 at regions other than entrance zone 335 or the constriction region 340 has a height of about 225 pm.

[0062] In some cases, the device 310 can include one or more valves (e.g., a first valve, a second value, a third valve, and so on), one or more valve actuators to operate the one or more valves, or one or more valve actuator vias to couple the valve actuators to the valves. FIG. 5D illustrates a valve, e.g., a valve with a flexible membrane, which can include a valve actuator configured to actuate the valve and a valve actuator via configured to operatively couple the valve to the valve actuator. Valves, valve actuators, and valve actuator vias can be designed, fabricated, or operated as described in Wang, Shaoxi, et al. “A review of capillary pressure control valves in microfluidics,” Biosensors 11.10 (2021): 405, which is incorporated by reference herein in its entirety. In some cases, the one or more valves may be coupled to the one or more valve actuators using one or more valve actuator vias. For example, operation of the one or more valves may include operations associated with increasing, reducing, restricting, or stopping the flow of the sample through the channel 330, the entrance zone 335, or the constriction region 340.Alternatively or additionally, operation of the one or more valves may include operations associated with extracting or removing the concentrated biomolecules from the device 310. For example, the valve actuator can be configured operate the valve 355 by generating pressure (e.g., pneumatic pressure, hydraulic pressure, or electrostatic pressure), which is transmitted to the valve 355 through the valve actuator via. In some cases, the operating of valve 355 can include fully opening or closing the valve 355 or partially opening or closing the valve 355. Partially opening or closing the valve 355 can include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the fully open or closed position of the valve 355.

[0063] In some cases, as illustrated in FIG. 5D, the valve 355 can be designed using a membrane to completely or partially open or close the valve 355. In some cases, the membrane can include a flexible membrane or a semirigid membrane. In some cases, the flexible membrane of the valve 355 may be located proximate to the channel 330, the entrance zone 335, or the constriction region 340. In some cases, the flexible membrane of the valve 355 may be located above, below, or to a side of the channel 330, the entrance zone 335, or the constriction region 340. In some cases, the flexible membrane of the valve 355 may be coupled to the channel 330, the entrance zone 335, or the constriction region 340. In some cases, the flexible membrane may be coupled to the valve actuator through the valve actuator via. In some cases, the flexible membrane can be fabricated from any material suitable for the operation of the valve 355. For example, the flexible membrane can be fabricated from rigid polymers or semirigid polymers such as polymethylmetacrylate (PMMA); from elastomers such as poly dimethyl siloxane (PDMS); from inorganics such as glass or silicon; or any combination thereof.

[0064] In some cases, the valve actuator can include a means for operating the valve 355. For example, valve actuator may use pneumatic actuation or switching, hydraulic actuation or switching, or electrical actuation or switching. Pneumatic actuation can include generating pneumatic pressure (e.g., air pressure) to operate the valve 355. Hydraulic actuation can include generating hydraulic pressure (e.g., fluid pressure) to operate the valve 355. Electrical actuation can include generating electrical pressure (e.g., electrostatic pressure) to operate the valve 355. In some cases, the pressure (e.g., pneumatic pressure, hydraulic pressure, or electrical pressure) may be transmitted from the valve actuator to the flexible membrane of valve 355 through the valve actuator via.

[0065] In some cases, the device 310 can include one or more ports configured for extracting the concentrated biomolecules from the device 310. For example, the extraction port or collection region 380 can be configured to receive the concentrated biomolecules from the device 310 or otherwise flow the concentrated biomolecules out of the device 310. In some embodiments, the collection region 380 is coupled to a collection device. In some embodiments, the collectiondevice is configured to extract concentrated nucleic acids from the collection region. For example, a syringe may be used as a collection device to receive the concentrated biomolecules from the device 310 or otherwise flow the concentrated biomolecules out of the device 310. In some embodiments, the device further comprises a third valve concentric with the constriction region 340 and configured to extract the concentrated nucleic acids from a collection region 380. In some cases, the concentrated biomolecules remain in the device 310 for further processing on the device 310.

[0066] In some cases, the concentrated biomolecules are moved to another region of the device 310 for further processing. For example, a second electric field may be generated to move the concentrated biomolecules to another region of the device 310 for further processing. In some cases, the second electric field can be the same electric field as the electric field for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the second electric field can be another electric field configured to move the concentrated biomolecules to another region of the device 310 for further processing. For example, the second electric field can be an AC or DC signal that is orthogonal or substantially orthogonal to the electric field for concentrating or extracting biomolecules. In some cases, the second electric field can be an AC or DC signal that is parallel to or substantially parallel to the electric field for concentrating or extracting biomolecules. In some cases, the second electric field generates an electrophoretic force to move the concentrated biomolecules to another region of the device 310 for further processing. In some cases, the second electric field generates a di electrophoretic force to move the concentrated biomolecules to another region of the device 310 for further processing. In some cases, the second electric field generates a combination of electrophoretic force and di electrophoretic force to move the concentrated biomolecules to another region of the device 310 for further processing.

[0067] In some cases, the device 310 may be fabricated from any suitable material compatible (e.g., biocompatible material) for performing one or more methods described herein. For example, suitable materials may be associated with materials used for microfluidic devices having one or more microchannels. In some embodiments, the channel 330 comprises a microchannel of a microfluidic device. In some embodiments, the microfluidic device comprises two or more instances of the microchannel each comprising a constriction region configured to extract nucleic acids from a sample. In some embodiments, the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass.

[0068] FIG. 4B depicts a closer view of the constriction region 340 of the channel 330. In some cases, the valve 355 may be co-located with or integrated into the channel 330, the entrance zone335, or the constriction region 340. In some cases, one or more corners of the constriction region 340 can be formed or fabricated with square comers or round corners. Round corners may help to reduce stagnation of the flow of the sample through the channel 330, the entrance zone 335, or the constriction region 340. Reducing stagnation can help to improve the concentrating or extracting of the biomolecules (e.g., nucleic acids or proteins) from biological samples. For example, round corners may reduce stagnation and improve concentrating or extracting of the biomolecules by at least about 10%, 20%, 30%, or more compared to a constriction region that uses square corners. In some cases, the corners may be formed or fabricated with a predetermined fillet radius. In some embodiments, the constriction region 340 comprises one or more comers each with a fillet radius ranging from about 0 pm to about 200 pm. In some embodiments, the constriction region 340 comprises one or more corners each with a fillet radius ranging from about 10 pm to about 100 pm. In some embodiments, the constriction region 340 comprises one or more corners each with a fillet radius ranging from about 20 pm to about 50 pm.

[0069] In some cases, the device 310 can include an electric field generator configured to generate an electric field along the channel 330. The electric field can generate an electrophoretic flow of the EH flow. In some embodiments, the electric field generator generates a first voltage at an electrode at a first position and a second voltage at an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region 340. In some embodiments, the electric field generated by the first and second voltages is substantially uniform along the length of the channel 330. For example, the electric field may comprise electric field lines that are substantially parallel to each other to thereby generate an electrophoretic force on some or all of the biomolecules of the sample. Alternatively or additionally, the electric field may comprise electric field lines that are substantially not parallel to each other (e.g., gradients in the electric field) to thereby generate a dielectrophoretic force on some or all of the biomolecules of the sample.

[0070] In some embodiments, the first and the second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow. In some embodiments, the first and second voltages are generated outside of the channel 330 and electrically coupled to the channel 330. For example, the device 310 may include an electric field generator (e.g., a signal generator and a signal amplifier) that is located off of the device 310 but is coupled to the channel 330. Coupling can include use of one or more electrical connectors located near the input 310 and the output port 320 that electrically couple to the channel 330 through the fluid of the sample. In some embodiments, the first and second voltages are generated proximate to the channel 330 and electrically coupled to the channel 330.

[0071] In some embodiments, the first and second voltages are generated at microelectrodes integrated into the channel 330 and electrically coupled to the channel 330. For example, the device 310 may include an electric field generator (e.g., a signal generator and a signal amplifier) that is located off of the device 310 and is coupled to the channel 330. Coupling can include use of one or more integrated microelectrodes located near the channel 330, the entrance zone 335, or the constriction region 340 that electrically couple to the channel 330 through contact with the fluid of the sample. In some cases, a dielectric layer separates the integrated microelectrodes from the fluid of the sample and couples to the channel 330, the entrance zone 335, or the constriction region 340 by noncontact with the fluid of the sample.

[0072] In some cases, the device 310 can include a fluid flow generator configured to generate a fluidic flow along the channel 330. The fluid flow generator can generate a convective flow of the EH flow. In some cases, the fluid field generator generates a first pressure at a first position and a second pressure at a second position, wherein the first position and the second position are located on opposite sides of the constriction region 340. In some cases, the pressure drop generated by the first and second pressures decreases uniformly along the length of the channel 330, the entrance zone 335, and the constriction region 340. In some cases, the pressure drop generated by the first and second pressures decreases nonuniformly along the length of the channel 330, the entrance zone 335, and the constriction region 340.

[0073] In some cases, the first and the second pressures are set to establish the convective flow that is opposite to the electrophoretic flow. In some cases, the first and second pressures are generated outside of the channel 330 and fluidically coupled to the channel 330. For example, the device 310 may include a fluid flow generator (e.g., a mechanical or electrical pump) that is located off of the device 310 but is coupled to the channel 330. Coupling can include use of one or more fluidic connectors located near the input 310 and the output port 320 that fluidically couple to the channel 330 through the fluid of the sample. In some cases, the first and second pressures are generated proximate to the channel 330 and fluidically couple to the channel 330. In some cases, the first and second pressures are generated at one or more positions integrated into the channel 330 and fluidically couple to the channel 330. Coupling can include use of one or more integrated micropumps located near the channel 330, the entrance zone 335, or the constriction region 340 that fluidically couple to the channel 330 through contact with the fluid of the sample.

[0074] FIG. 4C illustrates an example workflow for performing any one or more methods disclosed herein. For example, the workflow can include operations of generating an EH flow (e.g., turning on a fluid flow generator and an electric field generator, each not shown for clarity) through the channel 330; isolating the channel 330, the entrance zone 335, or the constrictionregion 340 by stopping the EH flow (e.g., turning off the fluid flow generator or the electric field generator, each not shown for clarity) of the sample or by actuating one or more valves (e.g., valve 355) to stop the convective flow of the sample; isolating the channel 330, the entrance zone 335, or the constriction region 340 by actuating one or more valves (e.g., valve 355); and extracting the concentrated biomolecules via the extraction port or collection region 380. FIG. 4C further depicts actual operation of the example workflow for performing any one or more methods disclosed herein.Description of devices such as device 410

[0075] FIGs. 4A-4D depict different embodiments of a device 410 that can be configured to implement any one or more methods disclosed herein. The device 410 can include some or all features of device 310 and include additional features.

[0076] FIG. 4A illustrates a plan view of the device 410, which can have a nonlimiting footprint of 35 mm wide by 50 mm long. The device 410 can include an input port 410, an output port 420, an output port via 425, a channel 430, an entrance zone 435 of the channel 430, a constriction region 440 of the channel 430, a first valve actuator (not shown), a valve 455, a first valve actuator via (not shown), an extraction port or collection region 480, or an extraction port via 485. The device 410 can further include additional features such as a second valve actuator (not shown), a valve 465, and a second valve actuator via (not shown). Generally, the device 410 can be included in a cartridge assembly in a similar manner as cartridge assembly 300, described herein elsewhere. Generally, the valve actuators, valves, and valve actuator vias of device 410 can be fabricated and operate in a similar or same manner as the valve actuators, valves, and valve actuator vias of device 310. FIG. 5D illustrates a valve, e.g., a valve with a flexible membrane, which can include a valve actuator configured to actuate the valve and a valve actuator via configured to operatively couple the valve to the valve actuator. Valves, valve actuators, and valve actuator vias can be designed, fabricated, or operated as described in Wang, Shaoxi, et al. “A review of capillary pressure control valves in microfluidics,” Biosensors 11.10 (2021): 405, which is incorporated by reference herein in its entirety.

[0077] In contrast to device 310, device 410 may include the second valve actuator, the valve 465, and the second valve actuator via configured to stop the fluidic flow of the sample through the channel 430, the entrance zone 435 of the channel 430, and the constriction region 440 of the channel 430. Also, the second valve actuator, the valve 465, and the second valve actuator via may be configured to isolate the concentrated biomolecules in the entrance zone 435, the constriction region 440, or the channel 430.

[0078] In some cases, the device 410 can include an electric field generator configured to generate an electric field along the channel 430. The electric field can generate an electrophoretic flow of the EH flow in a similar or same manner as device 310 or device 510.

[0079] In some cases, the device 410 can include a fluid flow generator configured to generate a fluidic flow along the channel 430. The fluid flow generator can generate a convective flow of the EH flow in a similar manner as device 310 or device 510.

[0080] FIG. 4B illustrates a closer view of the constriction region 440 of the channel 430. The constriction region 440 can be designed or operated in a similar manner as with device 310 or device 510, described herein elsewhere. FIG. 4C illustrates an example workflow for performing any one or more methods disclosed herein. The workflow can operate in a similar or same manner as with device 310 or device 510. FIG. 4D illustrates a length, a width, or a height of a channel, an entrance zone, and a constriction region.Description of devices such as device 510

[0081] FIG. 2B depicts an example cartridge assembly 300 that can be configured to implement any one or more methods disclosed herein. For example, the cartridge assembly 300 can include a cartridge holder 320 or a device, e.g., device 310, 410, or 510. The cartridge holder 320 may be configured to accept, retain, position, or align the device 510 to another device (e.g., equipment or instruments). In some cases, the cartridge holder 320 may be configured with a viewing window to view the device 510 from a position (e.g., above, below, or side) relative to the device 510. In some cases, the viewing can include optical viewing, infrared (IR) viewing, or ultraviolet (UV) viewing of biological samples in the device 510. In some cases, the cartridge holder 320 may be configured to reduce, suppress, or eliminate electromagnetic interference. In some cases, the device 510 may be configured to snap into the cartridge holder 520, plug and play into the cartridge holder 320, or fasten (e.g., screws) into the cartridge holder 320. In some cases, the cartridge holder 320 may be fabricated from a suitable material for the operation of device 510. For example, suitable materials can include plastics or metals that do not interfere with the operation of device 510.

[0082] In some cases, the device 510 may be electrically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include electrical connections associated with generating electric fields for EH flows, electrical connections associated with equipment or instruments to measure the concentrating or extracting of biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the device 510 may be mechanically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include mechanical connections associated with accepting, retaining, positioning, or aligning the device 510 to another device (e.g., equipment or instruments). In some cases, the device 510 may be fluidically coupled to thecartridge holder 320. For example, the cartridge holder 320 may include fluidic connections associated with generating fluidic flows for EH flows to concentrate or extract biomolecules from biological samples. In some cases, the device 510 may be pneumatically coupled to the cartridge holder 320. For example, the cartridge holder 320 may include pneumatic connections associated with actuating valves on the device 510 or generating fluidic flows for EH flows to concentrate or extract biomolecules from biological samples.

[0083] FIGs. 5A-5E depict different embodiments of the device 510 that can be configured to implement any one or more methods disclosed herein. The device 510 can include some or all features of device 310 or device 410 and include additional features.

[0084] FIG. 5A illustrates a plan view of the device 510, which can have a nonlimiting footprint of 35 mm wide by 60 mm long. The device 510 can include an input port 510, an input port via 515, an output port 520, an output port via 525, a channel 530, an entrance zone 535 of the channel 530, a constriction region 540 of the channel 530, a first valve actuator (not shown), a first and second valve 555, a first valve actuator via (not shown), a second valve actuator (not shown), a first and second valve 565, a second valve actuator via (not shown), an extraction port or collection region 580, or an extraction port via 585. Generally, the valve actuators, valves, and valve actuator vias of device 510 can be fabricated and operate in a similar or same manner as the valve actuators, valves, and valve actuator vias of device 310 or device 410.

[0085] In some cases, the device 510 can include the input port 510 configured to introduce the sample to the device 510 or otherwise flow the sample into the device 510. The input port 510 may be fluidically or structurally coupled to the entrance zone 535, the constriction region 540, the channel 530, and the output port 520. The device 510 can include the output port 520 configured to remove the sample from the device 510 or otherwise flow the sample out of the device 510. The output port 520 may be fluidically or structurally coupled to the entrance zone 535, the constriction region 540, the channel 530, and the input port 510. The device 510 can include the channel 530 configured to contain, seal, guide, or flow the sample from the input port 510 to the output port 520. The channel 530 may be fluidically or structurally coupled to the input port 510, the entrance zone 535, the constriction region 540, and the output port 520. The device 310 can include the channel 530 configured to contain, seal, guide, or flow the sample from the input port 510 to the output port 520. In some, cases, the channel 530 can include regions such as the entrance zone 535 or the constriction region 540. In some cases, the channel 530 may guide or flow the sample from the input port 510 to the output port 520 via the entrance zone 535 and the constriction region 540.

[0086] In some cases, the device 510 can include the entrance zone 535 configured to flow the sample to the constriction region 540 with a flow profile. The entrance zone 535 may be fluidicallyor structurally coupled to the input port 510, the constriction region 540, the channel 530, and the output port 520. In some cases, the entrance zone 535 may be used interchangeably with the constriction region 540. In some cases, as illustrated in FIG. 1C, the combination of the entrance zone 535 and the constriction region 540 may be designed to generate a fluidic flow profile (e.g., convective flow) with a flow speed or velocity gradient across or along the channel 530, the constriction region 540, or the channel 530. For example, the entrance zone 535 may be designed with a width, length, and height that is different than the width, length, or height of the constriction region 530 or of the channel 530. FIG. 5E illustrates the widths, lengths, and heights. The difference in the width, length, or height may generate a flow profile (e.g., the convective flow) that operates in combination with the electrophoretic flow to concentrate or extract biomolecules (e.g., nucleic acids or proteins) from biological samples near the channel 530, the entrance zone 535, or the constriction region 540, as described herein elsewhere. For example, the entrance zone 535 and the constriction region 540 may be designed with a shape that helps to optimize the generating of the fluidic flow profile. In some embodiments, an entrance zone 535 to the region comprising the constriction region 540 has a shape selected from square, rectangular, circular, hyperbolic, or semi-circular.

[0087] In some cases, the width, length, or height of the entrance zone 535 may be designed based at least on generating a ratio of the width, length, or height of the entrance zone 535 to a width, length, or height of the channel 530. In some cases, the width, length, or height may be designed based at least on generating a ratio of the width, length, or height of the constriction region 535 to a width, length, or height of the channel 530. In some embodiments, the region comprising the constriction region 540 of the channel 530 has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, llx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x wider than the channel 530.

[0088] In some cases, the entrance zone 535 may be designed with one or more widths, lengths, or heights. In some cases, the width, length, or height of the entrance zone 535 may include the width length, or height of the constriction region 540. In some cases, the width of the entrance zone 535 is from about 0.001 pm to about 100 mm. In some cases, the width of entrance zone 535 is about 1 mm. In some cases, the length of the entrance zone 535 is from about 0.001 pm to about 100 mm. In some cases, the length of the entrance zone 535 is about 10 mm. In some cases, the entrance zone 535 has a height ranging from about 0.001 nm to about 100 mm. In some cases, the entrance zone 535 has a height ranging from about 0.01 nm to about 10 mm. In some cases, the entrance zone 535 has a height ranging from about 0.1 nm to about 1 mm. In some cases, the entrance zone 535 has a height greater than 100 mm. In some cases, the entrance zone 535 has a height of about 225 pm.

[0089] In some cases, the device 510 can include the constriction region 540. The constriction region 540 may be fluidically or structurally coupled to the input port 510, the entrance zone 535, the channel 530, and the output port 520. In some cases, the constriction region 540 may be designed with one or more widths, lengths, or heights. In some cases, the width, length, or height of the constriction region 540 may include the width length, or height of the entrance zone 535. In some embodiments, a width of the constriction region 540 is from about 0.001 pm to about 100 mm. In some embodiments, a width of the constriction region 540 is about 1 mm. In some embodiments, a length of the channel 530 between an end of the constriction region 540 to an opposite end of the constriction region 540 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 530 between an end of the constriction region 540 to an opposite end of the constriction region 540 is about 10 mm. In some embodiments, the constriction region 540 has a height ranging from about 0.001 nm to about 100 mm. In some embodiments, the constriction region 540 has a height ranging from about 0.01 nm to about 10 mm. In some embodiments, the constriction region 540 has a height ranging from about 0.1 nm to about 1 mm. In some embodiments, the constriction region 540 has a height greater than 100 mm. In some embodiments, the constriction region 540 has a height of about 225 pm.

[0090] In some cases, the device 510 can include the channel 530 having one or more regions other than the entrance zone 535 or the constriction region 535. The one or more other regions may be fluidically or structurally coupled to the input port 510, the entrance zone 535, the constriction region 540, the channel 530, and the output port 520. In some cases, the one or more other regions of the channel 530 (e.g., regions other than the entrance zone 535 or the constriction region 540) may be designed with one or more widths, lengths, or heights. In some embodiments, a width of the channel 530 at regions other than the entrance zone 535 or the constriction region 540 is from about 0.001 pm to about 10 mm. In some embodiments, a width of the channel 530 at regions other than the entrance zone 535 or the constriction region 540 is about 1 mm. In some embodiments, a length of the channel 530 between the inlet port 510 and the constriction region 540 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 530 between the inlet port 510 and the constriction region 540 is about 10 mm. In some embodiments, a length of the channel 530 between an end of the constriction region 540 to the outlet port 520 is from about 0.001 pm to about 100 mm. In some embodiments, a length of the channel 530 between an end of the constriction region 540 to the outlet port 520 is about 20 mm. In some cases, the channel 530 at regions other than entrance zone 535 or the constriction region 540 has a height ranging from about 0.01 nm to about 10 mm. In some cases, the channel 530 at regions other than entrance zone 535 or the constriction region 540 has a height ranging from about 0.1 nm to about 1 mm. In some cases, the channel 530 at regions other than entrance zone 535 or the constrictionregion 540 has a height greater than 100 mm. In some cases, the channel 530 at regions other than entrance zone 535 or the constriction region 540 has a height of about 225 gm.

[0091] In some cases, the device 510 can include one or more valves (e.g., a first valve, a second value, a third valve, and so on), one or more valve actuators to operate the one or more valves, or one or more valve actuator vias to couple the valve actuators to the valves. In some cases, the one or more valves may be coupled to the one or more valve actuators using one or more valve actuator vias. FIG. 5D illustrates a valve, e.g., a valve with a flexible membrane, which can include a valve actuator configured to actuate the valve and a valve actuator via configured to operatively couple the valve to the valve actuator. Valves, valve actuators, and valve actuator vias can be designed, fabricated, or operated as described in Wang, Shaoxi, etal. “A review of capillary pressure control valves in microfluidics,” Biosensors 11.10 (2021): 405, which is incorporated by reference herein in its entirety.

[0092] In some cases, operation of the one or more valves may include operations associated with extracting or removing the concentrated biomolecules from the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the first and second valves 555 perform the operations associated with extracting or removing the concentrated biomolecules from the channel 530, the entrance zone 535, or the constriction region 540. In some embodiments, the first and second valves 555 are arranged to isolate the region comprising the constriction region 540. In some embodiments, the first and second valves 555 are arranged proximate to the region comprising the constriction region 540. In some embodiments, the first and second valves 555 isolate the region comprising the constriction region 540 and wherein the region contains a volume of about 10 gL.

[0093] In some cases, operation of the one or more valves may include operations associated with increasing, reducing, restricting, or stopping the flow of the sample through the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the third and fourth valves 565 perform the operations associated with increasing, reducing, restricting, or stopping the flow of the sample through the channel 530, the entrance zone 535, or the constriction region 540. In some embodiments, the device further comprises a third valve and a fourth valve 565, wherein the third valve and the fourth valve 565 are configured to stop the fluidic flow of the sample through the channel 530. In some cases, the third and fourth valves 565 are arranged to stop the fluidic flow of the sample through the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the third and fourth valves 565 are arranged proximate to the input port 510 or the output port 520.

[0094] In some cases, the first valve actuator can be configured operate the first and second valve 555 by generating pressure (e.g., pneumatic pressure, hydraulic pressure, or electrostaticpressure), which is transmitted to the first and second valve 555 through the second valve actuator via. In some cases, the operating of the first and second valve 555 can include fully opening or closing the first or second valve 555 or partially opening or closing the first or second valve 555. Partially opening or closing the first or second valve 555 can include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the fully open or closed position of the first or second valve 555.

[0095] In some cases, the second valve actuator can be configured operate the third and fourth valve 565 by generating pressure (e.g., pneumatic or hydraulic pressure), which is transmitted to the third and fourth valve 565 through the second valve actuator via. In some cases, the operating of the third and fourth valve 565 can include fully opening or closing the third or fourth valve 565 or partially opening or closing the third or fourth valve 565. Partially opening or closing the third or fourth valve 565 can include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the fully open or closed position of the third or fourth valve 565.

[0096] In some cases, as illustrated in FIG. 5D, the valves 555 or 565 can be designed using actuation of the membrane to completely or partially open or close the valves 555 or 565. In some cases, the membrane can include a flexible membrane or a semirigid membrane. In some cases, the flexible membrane of the valves 555 or 565 may be located proximate to the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the flexible membrane of the valves 555 or 565 may be located above, below, or to a side of the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the flexible membrane of the valves 555 or 565 may be coupled to the channel 530, the entrance zone 535, or the constriction region 540. In some cases, the flexible membrane may be coupled to the first and second valve actuators through the first and second valve actuator vias. In some cases, the flexible membrane can be fabricated from any material suitable for the operation of the valves 555 or 565. For example, the flexible membrane can be fabricated from rigid polymers or semirigid polymers such as polymethylmetacrylate (PMMA); from elastomers such as polydimethylsiloxane (PDMS); from inorganics such as glass or silicon; or any combination thereof.

[0097] In some cases, the first and second valve actuators can include a means for operating the valve 555 or 565. For example, first and second valve actuators may use pneumatic actuation or switching, hydraulic actuation or switching, or electrical actuation or switching. Pneumatic actuation can include generating pneumatic pressure (e.g., air pressure) to operate the valve 555 or 565. In some embodiments, the first and second valves 555 comprise a pneumatic valve. In some cases, the third and fourth valves 565 comprise a pneumatic valve. Hydraulic actuation can include generating hydraulic pressure (e.g., fluid pressure) to operate the valve 555 or 565. Electrical actuation can include generating electrical pressure (e.g., electrostatic pressure) to operate the valve 555 or 565. In some cases, the pressure (e.g., pneumatic pressure, hydraulicpressure, or electrical pressure) may be transmitted from the first and second valve actuator to the flexible membrane of valve 555 or 565 through the first and second valve actuator vias.

[0098] In some cases, the device 510 can include one or more ports configured for extracting the concentrated biomolecules from the device 510. For example, the extraction port or collection region 580 can be configured to receive the concentrated biomolecules from the device 510 or otherwise flow the concentrated biomolecules out of the device 510. In some embodiments, the collection region 580 is coupled to a collection device. In some embodiments, the collection device is configured to extract concentrated nucleic acids from the collection region 580. For example, a syringe may be used as a collection device to receive the concentrated biomolecules from the device 510 or otherwise flow the concentrated biomolecules out of the device 510. In some cases, the concentrated biomolecules remain in the device 510 for further processing on the device 510.

[0099] In some cases, the concentrated biomolecules are moved to another region of the device 510 for further processing. For example, a second electric field may be generated to move the concentrated biomolecules to another region of the device 510 for further processing. In some cases, the second electric field can be the same electric field as the electric field for concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the second electric field can be another electric field configured to move the concentrated biomolecules to another region of the device 510 for further processing. For example, the second electric field can be an AC or DC signal that is orthogonal or substantially orthogonal to the electric field for concentrating or extracting biomolecules. In some cases, the second electric field can be an AC or DC signal that is parallel to or substantially parallel to the electric field for concentrating or extracting biomolecules. In some cases, the second electric field generates an electrophoretic force to move the concentrated biomolecules to another region of the device 510 for further processing. In some cases, the second electric field generates a di electrophoretic force to move the concentrated biomolecules to another region of the device 510 for further processing. In some cases, the second electric field generates a combination of electrophoretic force and di electrophoretic force to move the concentrated biomolecules to another region of the device 510 for further processing.

[0100] In some cases, the device 510 may be fabricated from any suitable material compatible (e.g., biocompatible material) for performing one or more methods described herein. For example, suitable materials may be associated with materials used for microfluidic devices having one or more microchannels. In some embodiments, the channel 530 comprises a microchannel of a microfluidic device. In some embodiments, the microfluidic device comprises two or more instances of the microchannel each comprising a constriction region configured to extract nucleicacids from a sample. In some embodiments, the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass.

[0101] FIG. 5B illustrates a closer view of the constriction region 540 of the channel 530. In some cases, the valve 555 or 565 may be co-located with or integrated into the channel 530, the entrance zone 535, or the constriction region 540. In some cases, one or more corners of the constriction region 540 can be formed or fabricated with square corners or round corners. Round corners may help to reduce stagnation of the flow of the sample through the channel 530, the entrance zone 535, or the constriction region 540. Reducing stagnation can help to improve the concentrating or extracting of the biomolecules (e.g., nucleic acids or proteins) from biological samples. For example, round corners may reduce stagnation and improve concentrating or extracting of the biomolecules by at least about 10%, 20%, 30%, or more compared to a constriction region that uses square comers. In some cases, the corners may be formed or fabricated with a predetermined fillet radius. In some embodiments, the constriction region 540 comprises one or more corners each with a fillet radius ranging from about 0 pm to about 200 pm. In some embodiments, the constriction region 540 comprises one or more corners each with a fillet radius ranging from about 10 pm to about 100 pm. In some embodiments, the constriction region 540 comprises one or more corners each with a fillet radius ranging from about 20 pm to about 50 pm.

[0102] In some cases, the device 510 can include an electric field generator configured to generate an electric field along the channel 530. The electric field can generate an electrophoretic flow of the EH flow. In some embodiments, the electric field generator generates a first voltage at an electrode at a first position and a second voltage at an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region 540. In some embodiments, the electric field generated by the first and second voltages is substantially uniform along the length of the channel 530. For example, the electric field may comprise electric field lines that are substantially parallel to each other to thereby generate an electrophoretic force on some or all of the biomolecules of the sample. Alternatively or additionally, the electric field may comprise electric field lines that are substantially not parallel to each other (e.g., gradients in the electric field) to thereby generate a di electrophoretic force on some or all of the biomolecules of the sample.

[0103] In some embodiments, the first and the second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow. In some embodiments, the first and second voltages are generated outside of the channel 530 and electrically coupled to the channel 530. For example, the device 510 may include an electric field generator (e.g., a signal generator and signal amplifier) that is located off of the device 510 but is coupled to the channel 530. Coupling caninclude use of one or more electrical connectors located near the input 510 and the output port 520 that electrically couple to the channel 530 through the fluid of the sample. In some embodiments, the first and second voltages are generated proximate to the channel 530 and electrically coupled to the channel 530.

[0104] In some embodiments, the first and second voltages are generated at microelectrodes integrated into the channel 530 and electrically coupled to the channel 530. For example, the device 510 may include an electric field generator (e.g., a signal generator and signal amplifier) that is located off of the device 510 and is coupled to the channel 530. Coupling can include use of one or more integrated microelectrodes located near the channel 530, the entrance zone 535, or the constriction region 540 that electrically couple to the channel 530 through contact with the fluid of the sample. In some cases, a dielectric layer separates the integrated microelectrodes from the fluid of the sample and couples to the channel 530, the entrance zone 535, or the constriction region 540 by noncontact with the fluid of the sample.

[0105] In some cases, the device 510 can include a fluid flow generator configured to generate a fluidic flow along the channel 530. The fluid flow generator can generate a convective flow of the EH flow. In some cases, the fluid field generator generates a first pressure at a first position and a second pressure at a second position, wherein the first position and the second position are located on opposite sides of the constriction region 540. In some cases, the pressure drop generated by the first and second pressures decreases uniformly along the length of the channel 530, the entrance zone 535, and the constriction region 540. In some cases, the pressure drop generated by the first and second pressures decreases nonuniformly along the length of the channel 530, the entrance zone 535, and the constriction region 540.

[0106] In some cases, the first and the second pressures are set to establish the convective flow that is opposite to the electrophoretic flow. In some cases, the first and second pressures are generated outside of the channel 530 and fluidically coupled to the channel 530. For example, the device 510 may include a fluid flow generator (e.g., a mechanical or electrical pump) that is located off of the device 510 but is coupled to the channel 530. Coupling can include use of one or more fluidic connectors located near the input 510 and the output port 520 that fluidically couple to the channel 530 through the fluid of the sample. In some cases, the first and second pressures are generated proximate to the channel 530 and fluidically couple to the channel 530. In some cases, the first and second pressures are generated at one or more positions integrated into the channel 530 and fluidically couple to the channel 530. Coupling can include use of one or more integrated micropumps located near the channel 530, the entrance zone 535, or the constriction region 540 that fluidically couple to the channel 530 through contact with the fluid of the sample.

[0107] FIG. 5C illustrates an example workflow for performing any one or more methods disclosed herein. The workflow can operate in a similar or same manner as with device 310 or device 410. In contrast to device 310 or device 410, device 510 can use the second valve actuator, the third and fourth valve 565, and the second valve actuator via to stop the fluidic flow of the sample through the channel 530, the entrance zone 535 of the channel 530, or the constriction region 540 of the channel 530. Also, in contrast to device 310 or 410, device 510 can use the first valve actuator, the first and second valves 555, and the first valve actuator via to isolate the concentrated biomolecules in the entrance zone 535, the constriction region 540, or the channel 530. FIG. 5C further depicts actual operation of the example workflow for performing any one or more methods disclosed herein. FIG. 5D illustrates an example of a valve for performing any one or more methods disclosed herein in similar or same manner as device 310 or device 410.Description of devices such as high-throughput devices

[0108] FIG. 9 depicts a non-limiting example of a high-throughput device (HTD) for concentrating, preparing and / or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples, which can include certain inputs and outputs. Preparing can include preparing a library (or library prep) for sequencing. The HTD can include some or all features of device 310, device 410, or device 510, or additional features. The HTD can be configured to implement any one or more methods disclosed herein. In some cases, the HTD can be a scaled-up instance of device 310, device 410, or device 510. For example, compared to devices 310, 410, or 510, the HTD can extract volumes of at least 10 microliters (pL), 100 pL, 200 pL, 300 pL, and increments therein, or greater. For example, compared to devices 310, 410, or 510, the HTD can be configured with an extraction area of at least 10 square millimeters (mm2), 100 mm2, 200 mm2, 300 mm2, 400 mm2, 500 mm2, 600 mm2, 700 mm2, and increments therein, or greater. For example, compared to devices 310, 410, or 510, the HTD can include a channel depth of at least 100 micrometers (pm), 200 pm, 300 pm, 400 pm, 500 pm, and increments therein, or greater.

[0109] Additional features of the HTD can include on-device features for introducing cells and reagents to the HTD. Onboard reagents can include lysing reagents, fragmentation reagents, Proteinase K reagents, adapter reagents, and the like. Lysing reagents can be stored at room temperature and pre-installed on the HTD thereby streamlining methods herein and reducing user time and effort. By including the lysing step as a feature on the HTD, methods herein can be end- to-end, e.g., beginning from raw cell samples to a prepared library, or plug and play, e.g., functionally inserted into an instrument. Channels of the HTD can include a network of channels (not shown). The network of channels can include channels and features for transferring, mixing, heating, disposing waste, and the like.Description of devices such as multiplexed devices

[0110] FIGs. 10A-10B depict a non-limiting example of a multiplexed device (MPD) for concentrating, preparing, and / or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. Preparing can include preparing a library (or library prep) for sequencing. Multiple channels of the MPD can be useful for separately processing different, separate samples independently thereby allowing multiple and different simultaneous purifications and extractions of different nucleic acids from different biological samples. FIG. 10A illustrates a top view of the MPD, which can include certain inputs and outputs. The MPD can include some or all features of device 310, device 410, or device 510, or additional features. The MPD can be configured to implement any one or more methods disclosed herein. In some cases, the MPD can be the same scale as device 310, device 410, or device 510 but with a plurality of instances of device 310, device 410, or device 510. For example, compared to a single instance of device 310, 410, or 510, the MPD can extract volumes of at least 10 microliters (pL), 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, and increments therein, or greater. For example, compared to a single instance of device 310, 410, or 510, the MPD can be configured with an extraction area of at least 10 square millimeters (mm2), 50 mm2, 100 mm2, 150 mm2, 200 mm2, and increments therein, or greater. For example, compared to a single instance of device 310, 410, or 510, the MPD can include a channel depth of at least 100 micrometers (pm), 200 pm, 300 pm, 400 pm, and increments therein, or greater.[OHl] Additional features of the MPD can include on-device features for introducing cells and reagents to the MPD. Onboard reagents can include lysing reagents, fragmentation reagents, Proteinase K reagents, adapter reagents, and the like. Lysing reagents can be stored at room temperature and pre-installed on the MPD thereby streamlining methods herein and reducing user time and effort. By including the lysing step as a feature on the MPD, methods herein can be end- to-end, e.g., beginning from raw cell samples to a prepared library, or plug and play, e.g., functionally inserted into an instrument. Channels of the MPD can include a network of channels (not shown). The network of channels can include channels and features for transferring, mixing, heating, disposing waste, and the like. FIG. 10B illustrates different side views of different embodiments of the MPD, which can be configured with a plurality of levels z (or layers z) of differently sized arrays m by n. In some cases, the number of layers n of the MPD can include at least 1, 2, 3, 4, 5, 10, 25, 50, 100, and increments therein, or more. In some cases, the array of the MPD can be an m by n array. In some cases, the number m of the array can be at least 1, 2, 3, 4, 5, 10, 25, 50, 100, and increments therein, or more. In some cases, the number n of the array can be at least 1, 2, 3, 4, 5, 10, 25, 50, 100, and increments therein, or more.Description of devices using magnetic beads

[0112] Systems, methods, and devices herein can be used with magnetic beads for concentration (e.g., purification) and extraction of biomolecules, e.g., nucleic acids or proteins, from biological samples. In some cases, systems, methods, and devices herein can use any combination of magnetic beads and EHT for concentrating and extracting biomolecules. Using magnetic beads for concentrating and extracting biomolecules is known to those skilled in the art. Magnetic beadbased concentration and extraction can use magnetic beads coated with DNA-binding molecules. See, for example, Sheershika et al., “Advances in DNA Extraction Techniques: A Comprehensive Review of Methods and Applications.” Plant Cell Biotechnology and Molecular Biology, 25.5-6 (2024): 30-42, which is incorporated by reference herein in its entirety.

[0113] Unconventional and novel uses of magnetic beads described herein include at least using magnetic beads with systems, methods, and devices herein in continuous flow microfluidic channels for end-to-end workflows, e.g., plug and play manner. In some cases, devices for use with magnetic beads can include any device described herein. For example, devices can include some or all features of device 310, device 410, or device 510 herein, or additional features. For example, devices can include some or all features of the high-throughput device (HTD) herein, or additional features. For example, devices can include some or all features of the multiplexed device (MTD) herein, or additional features. In some cases, devices can be configured to implement any one or more methods disclosed herein. For example, devices can be configured to use magnetic beads in a continuous microfluidic flow format to implement any one or more methods disclosed herein.

[0114] In some cases, devices 310, 410, 510, the HTD, or the MTD herein can be modified for use with magnetic beads to concentrate and extract biomolecules, e.g., nucleic acids or proteins, from biological samples. As a nonlimiting example using device 510 depicted in FIG. 5A, device 510 can be modified by modifying any feature of device 510. For example, the width, length, or height of channel 530 can be modified. For example, the width, length, or height of entrance zone 535 can be modified. For example, the width, length, or height of constriction region 540 can be modified. For example, any combination of the width, length, or height of channel 530, entrance zone 535, and constriction region 540 can be modified. As but one example, the width of entrance zone 535 can be the same width as the width of channel 530 thereby establishing a single continuous channel having the same width from an input port to an output port. In some cases, the ratio of the width, length, or height of entrance zone 535 to the width, length, or height of channel 530 can be modified. As but one example, the ratio can of the width can be a 1 : 1 ratio thereby establishing a single continuous channel having the same width from an input port to an output port.

[0115] In some cases, systems, methods, and devices can be configured to use magnetic fields for establishing magnetic forces on magnetic beads thereby concentrating and extracting biomolecules. In some cases, magnetic fields and forces thereof can be used in combination with electrohydrodynamic trapping (EHT) herein for concentrating and extracting biomolecules. Using magnetic fields to establish magnetic forces is known to those skilled in the art. Unconventional and novel uses of magnetic fields described herein include at least using magnetic fields with systems, methods, and devices herein in continuous flow microfluidic channels for end-to-end workflows, e.g., plug and play manner. As but one example, a workflow can include performing: (i) cell lysation, (ii) purification of DNA using magnetic beads and / or EHT trapping, (iii) library preparation, (iv) clean-up of DNA using magnetic beads and / or EHT trapping, and (v) extraction of the purified DNA.

[0116] For example, magnetic field forces and / or electric field forces herein can be used to concentrate and extract biomolecules from biological samples. As but one example, systems, methods, and devices herein which use magnetic fields and / or electric fields can be used to replace or perform certain steps of other workflows for concentrating and extracting biological samples. For example, as described in Examples herein, systems, methods, and devices herein can be configured to replace and perform steps of workflows performed by the Oxford Nanopore® (ONT) Ultra-Long DNA Sequencing Kit or the Oxford Nanopore® Rapid Barcoding Kit. For example, a workflow can include performing: (i) cell lysation, (ii) purification of DNA using magnetic beads and / or EHT trapping, (iii) library preparation, (iv) clean-up of DNA using magnetic beads and / or EHT trapping, and (v) extraction of the purified DNA. Systems, methods, and devices herein can replace at least steps (ii) and (iv) of the workflow. In some cases, systems, methods, and devices herein can replace steps (i) - (v).Description of devices using surface binding

[0117] Systems, methods, and devices herein can be used without magnetic beads or EHT for concentration (e.g., purification) and extraction of biomolecules, e.g., nucleic acids or proteins, from biological samples. In some cases, systems, methods, and devices herein can use any combination of surface binding, magnetic beads, and EHT for concentrating and extracting biomolecules. Using surface binding of DNA for concentrating and extracting biomolecules is known to those skilled in the art. See, for example, Wen, et al., “Purification of nucleic acids in microfluidic devices.” Analytical chemistry 80.17 (2008): 6472-6479, which is incorporated by reference herein in its entirety.

[0118] Unconventional and novel uses of surface binding described herein include at least using surface binding with systems, methods, and devices herein in continuous flow microfluidic channels for end-to-end workflows, e.g., plug and play manner. In some cases, devices for usewith surface binding can include any device described herein. For example, devices can include some or all features of device 310, device 410, or device 510 herein, or additional features. For example, devices can include some or all features of the high-throughput device (HTD) herein, or additional features. For example, devices can include some or all features of the multiplexed device (MTD) herein, or additional features. In some cases, devices can be configured to implement any one or more methods disclosed herein. For example, devices can be configured to use surface binding in a continuous microfluidic flow format to implement any one or more methods disclosed herein.

[0119] In some cases, devices 310, 410, 510, the HTD, or the MTD herein can be modified for use with surface binding to concentrate and extract biomolecules, e.g., nucleic acids or proteins, from biological samples. As a nonlimiting example using device 510 depicted in FIG. 5A, device 510 can be modified by modifying any surface or feature of device 510. For example, any surface of device 510, e.g., channel 530, entrance zone 535, and constriction region 540, can be modified to concentrate or extract biomolecules. For example, the width, length, or height of channel 530 can be modified. For example, the width, length, or height of entrance zone 535 can be modified. For example, the width, length, or height of constriction region 540 can be modified. For example, any combination of the width, length, or height of channel 530, entrance zone 535, and constriction region 540 can be modified. As but one example, the width of entrance zone 535 can be the same width as the width of channel 530 thereby establishing a single continuous channel having the same width from an input port to an output port. In some cases, the ratio of the width, length, or height of entrance zone 535 to the width, length, or height of channel 530 can be modified. As but one example, the ratio can of the width can be a 1 : 1 ratio thereby establishing a single continuous channel having the same width from an input port to an output port.

[0120] In some cases, surface binding, magnetic field forces, and / or electric field forces herein can be used to concentrate and extract biomolecules from biological samples. As but one example, systems, methods, and devices herein which use surface binding, magnetic fields, and / or electric fields can be used to replace or perform certain steps of other workflows for concentrating and extracting biological samples. For example, as described in Examples herein, systems, methods, and devices herein can be configured to replace and perform steps of workflows performed by the Oxford Nanopore® (ONT) Ultra-Long DNA Sequencing Kit or the Oxford Nanopore® Rapid Barcoding Kit. For example, a workflow can include performing: (i) cell lysation, (ii) purification of DNA using surface binding, magnetic beads, and / or EHT trapping, (iii) library preparation, (iv) clean-up of DNA using surface binding, magnetic beads, and / or EHT trapping, and (v) extraction of the purified DNA. Systems, methods, and devices herein can replace at least steps (ii) and (iv) of the workflow. In some cases, systems, methods, and devices herein can replace steps (i) - (v).Systems for extracting biomolecules from biological samples

[0121] Systems can include devices disclosed herein, which can be configured to implement methods disclosed herein to extract biological molecules (e.g., nucleic acids or proteins) from fluid samples (e.g., biological samples), in accordance with some embodiments. In some cases, a sample can comprise a fluid sample. In some cases, the fluid sample can comprise one or more types of biological molecules. In some cases, the one or more types of biological molecules can comprise nucleic acids, deoxyribonucleic acids (DNA), ribonucleic acids (RNA), proteins, or any combination thereof. In some cases, a volume of the fluid sample can include a range of about 1 nanoliter (nL) to about 10 milliliters (mL). In some embodiments, the sample comprises the nucleic acids. In some embodiments, the sample comprises proteins. In some embodiments, the sample comprises a cell lysate sample. In some embodiments, the sample comprises a biological fluid. In some embodiments, the biological fluid comprises cellular nucleic acids. In some embodiments, the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample. In some embodiments, the sample comprises a buffer. In some embodiments, the buffer has an ionic concentration of about 0 mM to about 100 mM. In some embodiments, the buffer has an ionic concentration of greater than about 100 mM. In some embodiments, the buffer has a pH ranging from about 6.5 to about 8.5. In some embodiments, the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof.

[0122] FIGs. 2A-2C depict an example system that can include equipment, instruments, or devices configured to implement methods disclosed herein. For example, as depicted in FIG. 2A, equipment or instruments can include electric field generators (e.g., signal generators and signal amplifiers), oscilloscopes, signal analyzers, spectrum analyzers, power analyzers, ammeters, voltmeters, calorimeters, fluid flow generators (mechanical pumps or electric pumps), fluid flow meters, temperature recorders, manometers, data recorders, data analyzers, computing systems, microscopes (e.g., optical microscopes or fluorescent microscopes), interferometers, PCR machines, short-read DNA sequencers, long-read DNA sequencers, mass spectrometers, nuclear magnetic resonance (NMR) spectrometers, and the like. FIG. 2B depicts a device (e.g., device 310) disclosed herein elsewhere that can be integrated into the system for extracting biomolecules (e.g., nucleic acids or proteins) from a sample. In some cases, the device can be configured to insert into a cartridge assembly, e.g., cartridge assembly 300, which can include a cartridge holder, e.g., cartridge holder 320. FIG. 2C depicts an example device that can include the device (e.g., device 310, 410, 510, the HTD, or the MPD) in a stack of other devices such as a heat sink, a controller, a thermal pad, a metal sheet, and a thermal cycler (e.g., a thermostat controller and thermocouples).

[0123] FIGs. 6A-6B and 7A-7B illustrate that devices disclosed herein can be integrated with other devices configured to perform one or more other operations associated with concentrating or extracting biomolecules (e.g., nucleic acids or proteins) from biological samples. In some cases, the one or more operations can be performed before, during, or after methods disclose herein. In some cases, the one or more other operations can include operations associated with polymerase chain reactions (PCR) of a sample. For example, operations can include mixing a sample before, during, or after a PCR of the sample. In some embodiments, the device further comprises an element configured to mix the sample, wherein the element is integrated into the device or is coupled to the device. For example, operations can include a controller 620 configured to control a temperature of a thermal pad 630 before, during, or after PCR of the sample. In some embodiments, the device further comprises an element configured to heat the sample, wherein the element is integrated into the device or is coupled to the device. For example, operations can include a heat sink 610 configured to assist in controlling heat transfer to or from the device (e.g., device 310, 410, 510, the HTD, or the MPD) before, during, or after PCR of a sample. For example, operations can include a metal sheet 640 configured to also assist in controlling heat transfer to or from the device or to protect the device from temperatures that may be outside of operating temperatures for the device (e.g., device 310, 410, 510, the HTD, or the MPD) or for PCR protocols. For example, operations can include purifying the sample before, during, or after PCR of a sample. In some embodiments, the device further comprises an element configured to purify the sample, wherein the element is integrated into the device or is coupled to the device.

[0124] In some cases, the other devices can be integrated as a stack with devices disclosed herein, e.g., device 310, 410, 510, the HTD, or the MPD. For example, as illustrated in FIG. 6A, devices disclosed herein (e.g., device 310, 410, 510, the HTD, or the MPD) can be included on a bottom layer of the stack of other devices. Other devices can include heat sink 610, controller 620, thermal pad 630, or metal sheet 640. FIG. 6B further illustrates that other devices can include thermostat controller 650 and thermocouple 660 for cycling (e.g., thermal cycling) a temperate in the device 310, 410, 510, the HTD, or the MPD e.g., inside the channel of the cartridge. In some cases, devices herein can be fastened to (e.g., locked to) the other devices. The thermostat controller 650 can be configured to control or function as a thermoelectric cooler (TEC). For example, as illustrated in FIG. 7A, devices disclosed herein (e.g., device 310, 410, 510, the HTD, or the MPD) can be included on a top layer of the stack. For example, devices disclosed herein (e.g., device 310, 410, or 510) can be included as any layer of the stack. Other devices can include heat sink 610, controller 620, thermal pad 630, or metal sheet 640. FIG. 7B further illustrates that other devices can include thermostat controller 650 and thermocouple 660 for cycling (e.g., thermal cycling) a temperature in the device 310, 410, 510, the HTD, or the MPD, e.g., inside the channelof the cartridge. In some cases, devices herein can be fastened to (e.g., locked to) the other devices. The thermostat controller 650 can be configured to control or function as a thermoelectric cooler (TEC).Examples

[0125] While various examples of the present disclosure have been shown and described herein, such examples are provided by way of example only. Numerous variations, changes, or substitutions may occur without departing from the present disclosure. It should be understood that various alternatives to the examples described herein may be employed.

[0126] Systems, methods, and devices herein can provide improvements over typical approaches for preparing DNA for sequencing, as summarized in Table 1, and demonstrated by the Examples herein.Table 1Example 1 — Purifying and extracting DNA from biological samples

[0127] FIGs. 11A-11F illustrate results of purifying and extracting DNA from biological samples using systems, methods, and devices herein, in accordance with some embodiments. Systems, methods, and devices herein were demonstrated for purification and extraction of biological samples, e.g., DNA. A nonlimiting workflow included: (i) transfer cell aliquots to low bind tubes, (ii) add lysis buffer with Proteinase K (optional), (iii) (optionally) dilute with 0-10 mL of IX Tris- EDTA and mix gently using a wide bore pipette tip, (iv) insert solution into sample reservoir with pipette, and (v) begin protocols for mixing, heating, trapping, and extraction using systems, methods, and devices herein. The protocols can include: mixing the sample with lysis; heating thesample for 0-90 minutes at 0-100° Celsius (C); trapping to purify the DNA from the lysate; and extracting the purified the DNA. In some cases, heating can be performed for 10 minutes at 56°C.

[0128] FIG. 11A illustrates that purification and extraction using systems, methods, and devices herein does not statistically affect length distribution of DNA thereby demonstrating readiness for sequencing of DNA. In FIG. 11 A, the columns of the gel electrophoresis depict different samples, e.g., 21 pL lysate of a biological sample, for determining length distributions of the different samples. Columns labeled with “extracted” indicate the samples obtained using systems, methods, and devices herein while other columns indicate control samples. The rows indicate the distribution of DNA fragments of varying lengths. As demonstrated, systems, methods, and devices herein were able to obtain samples of uniformly long lengths of DNA, e.g., at least 1000 kilobase pairs (kbp), from an extracted sample of 7 pL.

[0129] FIG. 11B illustrates that purification and extraction using systems, methods, and devices herein do not affect length distribution of DNA thereby demonstrating readiness for sequencing of DNA. FIG. 11B demonstrates that systems, methods, and devices herein are gentle when processing k-DNA thereby allowing use for DNA sequencing. For example, length measurements determined from gel electrophoresis are shown in FIG. 11B after multiple, repeated trapping events, e.g., up to 10 trappings or more. Each lane represents the number of times that input - DNA (e.g., 48.5 kbp) was trapped. As demonstrated in Table 2, the length of DNA is preserved after 10 or more trappings thereby demonstrating that less input sample is needed for use in DNA sequencing.Table 2

[0130] FIG. 11C illustrates that purification and extraction using systems, methods, and devices herein does not affect concentration of DNA thereby demonstrating readiness for sequencing of DNA. For example, as repeated in Table 3, systems, methods, and devices herein can retain at least 90% of k-DNA after multiple trappings.Table 3

[0131] FIG. HD illustrates purification and extraction performance compared to other methods, e.g., solid-phase extraction using magnetic beads. Compared to other methods, which can break down or degrade the integrity of DNA, systems, methods, and devices herein can preserve the length and integrity of DNA for sequencing. For example, FIG. 11D directly compares the length distribution of DNA following purification of a cellular lysate, using systems, methods, and devices herein, with magnetic bead approaches. The gel electrophoresis demonstrates less fragmentation of DNA than with magnetic bead approaches.

[0132] FIG. HE illustrates purification and extraction performance as compared to other methods, e.g., solid-phase extraction using magnetic beads, using the A260 / 280 ratio. The A260 / 280 ratio is the ratio of ultraviolet (UV) absorbances of a sample at 260 nanometers (nm) and 280 nm. The preferred test of purity, e.g., the A260 / 280 ratio, is whether samples can be sequenced by sequencing platforms. Here, samples were extracted following purification and extraction using systems, methods, and devices herein. Extracted samples were analyzed for purity using the A260 / 280 ratio. Solid-phase extraction methods (e.g., magnetic beads approaches) typically achieve a ratio of 1.8 to 2.0. As demonstrated in Table 4, systems, methods, and devices herein achieved a ratio of at least 1.70 thereby demonstrating readiness for sequencing of DNA.Table 4

[0133] FIG. HF illustrates that purification and extraction using systems, methods, and devices herein does not affect sequencing of DNA. As repeated in Table 5, systems, methods, and devices herein can maintain high quality scores. Quality scores (or Phred scores or q-scores) are a measureof the likelihood that a base call in DNA sequencing is incorrect. Quality scores are a metric used to assess the quality of sequencing data. Quality scores can be calculated using a negative log scale, where higher scores indicate greater confidence in the base call. A quality score of 20 means there is a 1 in 100 chance of an incorrect base call. For example, systems, methods, and devices herein achieved quality scores of at least 5+ (Q5) for 97.8% of reads of 378.2 Mbp, 7+ (Q7) for 88.1% of reads of 346.7 Mbp, 10+ (Q10) for 67.8% of reads of 282.6 Mbp, 12+ (Q12) for 12.3% of reads of 41.5 Mbp, and 15+ (QI 5) for 0.2% of reads of about 0 Mbp.Table 5Example 2 — Preparing DNA for ultra-long sequencing

[0134] FIGs. 12A-12E illustrate a non-limiting example workflow and results thereof for preparing DNA from biological samples for ultra-long sequencing using systems, methods, and devices herein. FIG. 12A illustrates the high-level workflow herein compared to other methods, e.g., Oxford Nanopore® (ONT) Ultra-Long DNA Sequencing Kit. Systems, methods, and devices herein were demonstrated for purification and clean-up of DNA for ultra-long sequencing of DNA. As illustrated in FIG. 12A, system, methods, and devices herein can replace certain steps used to prepare DNA for ultra-long sequencing thereby reducing time from 18 hours to 4 hours and reducing the number of steps from 55 steps to 6 steps. A nonlimiting workflow included: (i) transfer cell aliquots in low bind tubes, (ii) (optionally) dilute with 0-10 mL of IX Tris-EDTA and mix gently using a wide bore pipette tip, (iii) insert all reagents including library prep (e.g., fragmentation, adaptor, and lysis (+additive), (iv) insert solution into sample reservoir with pipette, and (v) begin protocols for mixing, heating, trapping, and extraction using systems, methods, and devices herein. The protocols can include: mixing sample with lysis; heating sample for 10 minutes at 56°C at 300 revolutions per minute (rpm)(or no shaking); trapping to purify high molecular weight DNA from lysate; flowing 250 microliters (pL) of MM1 ; gently mixing; heating sample by 30°C for 10 minutes, 75°C for 10 minutes; < 8°C for 10 minutes or more; adding 5 pL of Rapid Adapter® (RA) to sample and mixing; incubating for 30 minutes at room temperature, trapping to perform final clean-up of DNA; extracting the prepared DNA library sample for sequencing.

[0135] FIGs. 12B-12E demonstrate improved results using systems, methods, and devices herein compared to other methods, e.g., certain steps of Oxford Nanopore® (ONT) Ultra-Long DNA Sequencing Kit. In some cases, systems, methods, and devices were used to replace steps of purification, extraction, library prep, clean-up, and overnight elution. For example, as depicted in FIGs. 12B-12C (repeated in Tables 6 and 7), systems, methods, and devices herein can maintain high quality scores. For example, systems, methods, and devices herein achieved quality scores of at least 5+ (Q5) for 97.2% of reads of 2664.8 Mbp, 7+ (Q7) for 94.1% of reads of 2606.1 Mbp, 10+ (Q10) for 82.6% of reads of 2342.5 Mbp, 12+ (Q12) for 50.5% of reads of 1516.7 Mbp, and 15+ (Q15) for 0.1% of reads of about 0 Mbp. FIGs. 12D-12E compare ultra-long clean-up (purification) following attachment of the sequencing adapters. FIG. 12D compares the protocolperformance (e.g., time and effort) using the number of reads as a proxy, and FIG. 12E compares output for the two sets of samples. As demonstrated, performing clean-up using systems, methods, and devices herein can result in improvements, compared to the ONT Ultra-Long DNA Sequencing Kit, when performing long-read sequencing of DNA. For example, improvements can include reducing preparation time by at least 50%, naturally increasing throughput, reducing costs by at least 30% (e.g., by using less reagents, reducing workforce needs, and reducing plastic waste), reducing complexity by at least 90% (e.g. reducing the number of processing steps and labor hours), providing higher quality samples for long-read sequencing, and preparing longer samples and larger N50.Table 6Table 7

[0136] Further description of the workflow using the Oxford Nanopore® Technologies (ONT) Ultra-Long DNA Sequencing Kit is provided. Prior to loading DNA into the ONT sequencer and after purifying the DNA, it is necessary to attach sequencing adapters to the ends of the DNA. Once the adapters are attached, it is then necessary to perform another purification (or ‘clean-up’) to remove excess adapters, adapter dimers, and any other contaminants (e.g., enzymes). All steps in the workflow were performed using standard methods up to the final clean-up following adapter ligation. At this point, the sample was split in two. One half was purified using ONT’s approach (e.g., overnight separation) and the other was purified with systems, methods, and devices herein. As demonstrated by FIGs. 12B-12E, systems, methods, and devices herein can reduce processing time by 14 hours. The longer processing time using ONT’s approach results from ensuring that the DNA fully relaxes from any compacted or entangled states. Compared to the ONT approach, the gentle nature of systems, methods, and devices herein can reducing or eliminate entanglement of DNA. Compared to the ONT approach, systems, methods, and devices herein can reduce the number of steps. Compared to the ONT approach, systems, methods, and devices herein can improve sequencing metrics, e.g., higher quality DNA fragments and longer DNA fragments with a similar overall N50 (or a measure of median continuous sequence length).Example 3 — Preparing DNA for DNA barcoding

[0137] FIGs. 13A-13D illustrate a non-limiting example workflow and results thereof for preparing DNA from biological samples for DNA barcoding using systems, methods, and devices herein. FIG. 13A illustrates the high-level workflow herein compared to other methods, e.g., Oxford Nanopore® Rapid Barcoding Kit. Systems, methods, and devices herein were demonstrated for purification and clean-up of DNA for barcoding of DNA. As illustrated in FIG. 13A, system, methods, and devices herein can replace certain steps used to prepare DNA for barcoding thereby reducing time from 90 minutes to 45 minutes reducing the number of steps from 55 steps to 6 steps. A nonlimiting workflow included: (i) transfer cell aliquots in low bind tubes, (ii) (optionally) dilute with 0-10 mL of IX Tris-EDTA and mix gently using a wide bore pipette tip, (iii) insert all reagents including library prep Rapid Barcoding® Kit (RBK), (iv) insert solution into sample reservoir, and (v) begin protocols for mixing, heating, trapping, and extraction using systems, methods, and devices herein. The protocols can include: mixing samplewith lysis; heating sample for 10 minutes at 56°C with 300 rpm (or no shaking); trapping to purify DNA from lysate; introduce 1 pL of barcode; gently mixing; vi. heating sample by 30°C for 10 minutes; 80°C for 10 minutes; < 8°C for 10 minutes or more; trapping to perform final clean-up of DNA; adding 1 pL of Rapid Adapter® (RA) to sample and mixing; incubating for 5 minutes at room temperature; and extract the prepared DNA library sample for sequencing.

[0138] FIGs. 13B-13D demonstrate improved results of systems, methods, and devices herein compared to other methods, e.g., certain steps of Oxford Nanopore® Rapid Barcoding Kit. In some cases, systems, methods, and devices were used to replace steps of purification, extraction, tagmentation, clean-up, and adapter ligation. For example, as depicted in FIG. 13B (repeated in Table 8), systems, methods, and devices herein can maintain high quality scores. For example, systems, methods, and devices herein achieved quality scores of at least 5+ (Q5) for 99.6% of reads of 555.4 Mbp, 7+ (Q7) for 96.4% of reads of 545.2 Mbp, 10+ (Q10) for 86.7% of reads of 510.5 Mbp, 12+ (Q12) for 75.9% of reads of 461.2 Mbp, and 15+ (Q15) for 47.4% of reads of 316.3 Mbp. FIGs. 13C-13D compare ultra-long clean-up (purification) following attachment of the sequencing adapters. FIG. 13C compares the protocol performance (e.g., time and effort) using the number of reads as a proxy, and FIG. 13D compares output for the two sets of samples. As demonstrated, performing clean-up using systems, methods, and devices herein can result in improvements, compared to the Oxford Nanopore® Rapid Barcoding Kit, when performing sequencing of DNA. For example, improvements can include reducing preparation time by at least 50%, naturally increasing throughput, reducing costs by at least 30% (e.g., by using less reagents, reducing workforce needs, and reducing plastics), reducing complexity by at least 90% (e.g., reducing the number of processing steps and labor hours, providing higher quality samples for sequencing, and preparing longer samples and larger N50. Compared to the Oxford Nanopore® Rapid Barcoding Kit, systems, methods, and devices herein resulted higher concentrations of purified DNA. For example, starting with 89.2 nanograms per microliter (ng / pL), systems, methods, and devices herein obtained 14.4 ng / pL of DNA while the Rapid Barcoding Kit only obtained 2.12 ng / pL.Table 8

[0139] Further description of the workflow using the Oxford Nanopore® Rapid Barcoding Kit is provided. Cells were split and treated separately. One sample was purified using systems, methods, and devices herein while the other was purified using New England Biolabs® (NEB) Monarch high molecular weight (MW) DNA extraction kit. The samples were then handled using the same throughout, tagmentation process, and split again for clean-up. The samples were combined for the final adaptor ligation before sequencing together. As demonstrated by FIGs. 13C-13D, systems, methods, and devices herein can reduce preparation time by streamlining workflow processes. Compared to ONT’s barcoding approach, systems, methods, and devices herein can reduce costs due to more efficient use of resources and reagents. Compared to ONT’s barcoding approach, systems, methods, and devices herein can yield higher quality DNA fragments and improve the overall accuracy of sequencing results. Compared to ONT’s barcoding approach, systems, methods, and devices herein can yield higher N50 values thereby generating better assembly quality and longer contiguous sequences.Computing systems

[0140] In some cases, systems, methods, and devices disclosed herein may utilize one or more computing systems to perform operations associated with the systems, methods, or devices.

[0141] Referring to FIG. 8, a block diagram is shown depicting an exemplary machine that includes a computer system 800 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies for static code scheduling of the present disclosure. The components in FIG. 8 are examples only and do not limit the scope of use or functionality of any hardware,software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0142] Computer system 800 may include one or more processors 801, a memory 803, and a storage 808 that communicate with each other, and with other components, via a bus 840. The bus 840 may also link a display 832, one or more input devices 833 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 834, one or more storage devices 835, and various tangible storage media 836. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 840. For instance, the various tangible storage media 836 can interface with the bus 840 via storage medium interface 826. Computer system 800 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0143] Computer system 800 includes one or more processor(s) 801 (e.g., central processing units (CPUs) or general purpose graphics processing units (GPGPUs)) that carry out functions. Processor(s) 801 optionally contains a cache memory unit 802 for temporary local storage of instructions, data, or computer addresses. Processor(s) 801 are configured to assist in execution of computer readable instructions. Computer system 800 may provide functionality for the components depicted in FIG. 8 as a result of the processor(s) 801 executing non-transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 803, storage 808, storage devices 835, and / or storage medium 836. The computer-readable media may store software that implements particular embodiments, and processor(s) 801 may execute the software. Memory 803 may read the software from one or more other computer-readable media (such as mass storage device(s) 835, 836) or from one or more other sources through a suitable interface, such as network interface 820. The software may cause processor(s) 801 to carry out one or more processes or one or more steps of one or more processes described or illustrated herein. Carrying out such processes or steps may include defining data structures stored in memory 803 and modifying the data structures as directed by the software.

[0144] The memory 803 may include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 804) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phasechange random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 805), and any combinations thereof. ROM 805 may act to communicate data and instructions unidirectionally to processor(s) 801, and RAM 804 may act to communicate data and instructions bidirectionally with processor(s) 801. ROM 805 and RAM 804 may include any suitable tangible computer-readable media described below. In one example, a basic input / output system 806(BIOS), including basic routines that help to transfer information between elements within computer system 800, such as during start-up, may be stored in the memory 803.

[0145] Fixed storage 808 is connected bidirectionally to processor(s) 801, optionally through storage control unit 807. Fixed storage 808 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 808 may be used to store operating system 809, executable(s) 810, data 811, applications 812 (application programs), and the like. Storage 808 can also include an optical disk drive, a solid-state memory device (e.g., flash -based systems), or a combination of any of the above. Information in storage 808 may, in appropriate cases, be incorporated as virtual memory in memory 803.

[0146] In one example, storage device(s) 835 may be removably interfaced with computer system 800 (e.g., via an external port connector (not shown)) via a storage device interface 825. Particularly, storage device(s) 835 and an associated machine-readable medium may provide nonvolatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 800. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 835. In another example, software may reside, completely or partially, within processor(s) 801.

[0147] Bus 840 connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus 840 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.

[0148] Computer system 800 may also include an input device 833. In one example, a user of computer system 800 may enter commands and / or other information into computer system 800 via input device(s) 833. Examples of an input device(s) 833 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect®, Leap Motion®, or the like. Input device(s) 833 may be interfaced to bus 840via any of a variety of input interfaces 823 (e.g., input interface 823) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.

[0149] In particular embodiments, when computer system 800 is connected to network 830, computer system 800 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 830. Communications to and from computer system 800 may be sent through network interface 820. For example, network interface 820 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 830, and computer system 800 may store the incoming communications in memory 803 for processing. Computer system 800 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 803 and communicated to network 830 from network interface 820. Processor(s) 801 may access these communication packets stored in memory 803 for processing.

[0150] Examples of the network interface 820 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 830 or network segment 830 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 830, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0151] Information and data can be displayed through a display 832. Examples of a display 832 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 832 can interface to the processor(s) 801, memory 803, and fixed storage 808, as well as other devices, such as input device(s) 833, via the bus 840. The display 832 is linked to the bus 840 via a video interface 822, and transport of data between the display 832 and the bus 840 can be controlled via the graphics control 821. In some embodiments, the display is a video projector. In some embodiments, the display is a headmounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive®, Oculus Rift®, Samsung Gear VR®, Microsoft HoloLens®, Razer OSVR®, FOVE VR®, Zeiss VR One®, Avegant Glyph®, Freefly VR®headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.

[0152] In addition to a display 832, computer system 800 may include one or more other peripheral output devices 834 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 840 via an output interface 824. Examples of an output interface 824 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0153] In addition or as an alternative, computer system 800 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein. Reference to software in this present disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer- readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0154] Various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality.

[0155] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0156] The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, harddisk, a removable disk, a CD-ROM, or any other form of storage medium. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0157] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers, in various embodiments, include those with booklet, slate, and convertible configurations.

[0158] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Suitable server operating systems include, by way of non-limiting examples, FreeBSD®, OpenBSD®, NetBSD®, Linux®, Apple® Mac OS X Server®, Oracle Solaris®, Windows Server®, and Novell NetWare®. Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft Windows®, Apple Mac® OS X, UNIX®, and UNIX- like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia Symbian® OS, Apple® iOS, Research In Motion BlackBerry® OS, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile OS, Linux®, and Palm® WebOS. Suitable media streaming device operating systems include, by way of nonlimiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Suitable video game console operating systems include, by way of non-limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One®, Nintendo Wii®, Nintendo Wii U®, and Ouya®. Suitable virtual reality headset systems include, by way of non-limiting example, Meta Oculus®.Non-transitory computer readable storage mediums

[0159] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computingdevice. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In still further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semipermanently, or non-transitorily encoded on the media.Computer programs

[0160] In some cases, computer programs can be configured to implement any one or more methods disclosed herein. In another aspect, disclosed herein is a computer program product for extracting nucleic acids from a sample, the computer program product comprising at least one non-transitory computer-readable medium having computer-readable program code portions embodied therein, the computer-readable program code portions comprising: (1) an executable portion configured generate a fluidic flow of the sample through a channel, wherein the sample comprises the nucleic acids; (2) an executable portion configured to establish an electric field along a length of the channel, wherein the electric field is configured to generate an electrophoretic flow that is opposite to the fluidic flow; (3) an executable portion configured to concentrate the nucleic acids in a region comprising a constriction region of the channel, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; (4) an executable portion configured to isolate the region comprising the constriction region from other regions of the channel, wherein substantially all of the concentrated nucleic acids remain in the region; and (5) an executable portion configured to extract the nucleic acids from the region comprising the constriction region.

[0161] In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types. In light of the present disclosure provided herein, a computer program may be written in various versions of various languages.

[0162] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises onesequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.Terms and Definitions

[0163] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure belongs.

[0164] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0165] As used herein, the term “about” in some cases refers to an amount that is approximately the stated amount.

[0166] As used herein, the term “about” refers to an amount that is near the stated amount by 10%, 5%, or 1%, including increments therein.

[0167] As used herein, the term “about” in reference to a percentage refers to an amount that is greater or less the stated percentage by 10%, 5%, or 1%, including increments therein.

[0168] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0169] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. It is not intended that the present disclosure be limited by the specific examples provided within the specification. While the present disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions may occur without departing from the present disclosure. Furthermore, it shall be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing thepresent disclosure. It is therefore contemplated that the present disclosure shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present disclosure and that systems, methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for extracting nucleic acids from a sample, comprising: a) generating a fluidic flow of the sample through a channel, wherein the sample comprises the nucleic acids; b) establishing an electric field along a length of the channel, wherein the electric field is configured to generate an electrophoretic flow that is opposite to the fluidic flow; c) concentrating the nucleic acids in a region comprising a constriction region of the channel, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force concentrating the nucleic acids thereby yielding concentrated nucleic acids; d) isolating the region comprising the constriction region from other regions of the channel, wherein substantially all of the concentrated nucleic acids remain in the region; and e) extracting the nucleic acids from the region comprising the constriction region.

2. The method of claim 1, wherein the isolating in (d) further comprises stopping the fluidic flow of the sample through the channel.

3. The method of claim 2, wherein the stopping comprises closing valves to isolate the concentrated nucleic acids.

4. The method of claim 3, wherein the valves comprise a pneumatic valve.

5. The method of claim 3, wherein the valves are arranged to isolate the region comprising the constriction region.

6. The method of claim 3, wherein the valves are arranged proximate to the region comprising constriction region.

7. The method of claim 3, wherein the valves isolate the region comprising the constriction region and wherein the region contains a volume of about 10 pL.

8. The method of claim 1, wherein the region comprising the constriction region consists essentially of a subsection of the channel comprising the restriction region.

9. The method of claim 1, wherein the region comprising the constriction region: a) consists essentially of a subsection of the channel comprising the restriction region; and b) is isolatable from a remaining portion of the channel.

10. The method of claim 1, wherein the region comprising the constriction region: a) consists essentially of a subsection of the channel comprising the restriction region; and b) is isolatable from a remaining portion of the channel by a means comprising one or more valves.

11. The method of claim 1, wherein the extracting in (e) further comprises using pressure to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel.

12. The method of claim 11, wherein the force is effected by applying pneumatic pressure to the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction region and into the collection region.

13. The method of claim 1, wherein the extracting in (e) further comprises using an electrical force to force the concentrated nucleic acids to flow out of the region comprising the constriction region and into a collection region that is proximate to the channel.

14. The method of claim 13, wherein the electrical force is effected by applying an electrical field across or along the channel, causing the concentrated nucleic acids to flow out of the region comprising the constriction region and into the collection region.

15. The method of claim 11, further comprising coupling a collection device to the collection region, wherein the collection device is configured to extract the concentrated nucleic acids from the collection region.

16. The method of claim 1, wherein the sample comprises the nucleic acids.

17. The method of claim 1, wherein the sample comprises proteins.

18. The method of claim 16, wherein the sample comprises a cell lysate sample.

19. The method of claim 16, wherein the sample comprises a biological fluid.

20. The method of claim 19, wherein the biological fluid comprises cellular nucleic acids.

21. The method of claim 19, wherein the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample.

22. The method of claim 16, wherein the sample comprises a buffer.

23. The method of claim 22, wherein the buffer has an ionic concentration of about 0 mM to about 100 mM.

24. The method of claim 22, wherein the buffer has an ionic concentration of greater than about 100 mM.

25. The method of claim 22, wherein the buffer has a pH ranging from about 6.5 to about 8.5.

26. The method of claim 1, wherein the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof.

27. The method of claim 1, wherein the channel comprises a microchannel of a microfluidic device.

28. The method of claim 27, wherein the microfluidic device comprises two or more instances of the microchannel, each comprising a constriction region configured to extract nucleic acids from a sample.

29. The method of claim 27, wherein the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass.

30. The method of claim 1, wherein the fluidic flow of the sample through the channel is at a volumetric flow rate of from about 0.001 pL / min to about 1000 pL / min.

31. The method of claim 1, wherein the fluidic flow of the sample through the channel is at a flow speed of from about 0.001 mm / s to about 500 mm / s.

32. The method of claim 1, wherein the electric field has a field strength of from about 0.001 mV / cm to about 10 kV / cm.

33. The method of claim 1, wherein: a) the electric field has a field strength; b) the fluidic flow has a volumetric flow rate; and c) the field strength and the volumetric flow rate establish a field strength: volumetric flow rate ratio of about 4 V / cm: 1 pL / min.

34. The method of claim 1, wherein the electrohydrodynamic force causes the nucleic acids to substantially concentrate in an outer region of the region comprising the constriction region.

35. The method of claim 1, wherein the electrohydrodynamic force causes the nucleic acids to substantially concentrate at a top side portion, a bottom side portion, a left side portion, a right side portion, or any combination thereof of the region comprising the constriction region.

36. The method of claim 1, wherein the region comprising the constriction region of the channel has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x wider than the channel.

37. The method of claim 1, wherein the constriction region has a height ranging from about 0.001 nm to about 100 mm.

38. The method of claim 1, wherein the constriction region has a height ranging from about 0.01 nm to about 10 mm.

39. The method of claim 1, wherein the constriction region has a height ranging from about 0.1 nm to about 1 mm.

40. The method of claim 1, wherein the constriction region has a height greater than 100 mm.

41. The method of claim 1, wherein the constriction region has a height of about 225 pm.

42. The method of claim 1, wherein the constriction region comprises one or more corner regions each with a fillet radius ranging from about 0 pm to about 200 pm.

43. The method of claim 1, wherein the constriction region comprises one or more corner regions each with a fillet radius ranging from about 10 pm to about 100 pm.

44. The method of claim 1, wherein the constriction region comprises one or more corner regions each with a fillet radius ranging from about 20 pm to about 50 pm.

45. The method of claim 1, wherein an entrance zone to the region comprising the constriction region has a shape selected from square, rectangular, circular, hyperbolic, or semi-circular.

46. The method of claim 1, wherein the concentrated nucleic acids have an average length greater than about 300 kb.

47. The method of claim 1, wherein the concentrated nucleic acids have an average length greater than about 500 kb.

48. The method of claim 1, further comprising analyzing a portion or substantially all of the concentrated nucleic acids.

49. The method of claim 1, further comprising sequencing a portion or substantially all of the concentrated nucleic acids.

50. The method of claim 1, wherein the establishing in (b) further comprises: a) applying a first voltage to an electrode at a first position; and b) applying a second voltage to an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region, and wherein the electric field generated by the first and second voltages is substantially uniform along the length of the channel.

51. The method of claim 50, wherein the first and second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow.

52. The method of claim 50, wherein the first and second voltages are generated outside of the channel and electrically coupled to the channel.

53. The method of claim 50, wherein the first and second voltages are generated proximate to the channel and electrically coupled to the channel.

54. The method of claim 50, wherein the electrodes comprise microelectrodes integrated into the channel and electrically coupled to the channel.

55. The method of claim 1, further comprising heating the sample prior to or after the extracting in (e).

56. The method of claim 1, further comprising mixing the sample prior to or after the extracting in (e).

57. The method of claim 1, further comprising performing size selection on the sample prior to or after the extracting in (e).

58. The method of claim 1, further comprising purifying the sample prior to or after the extracting in (e).

59. A device for extracting nucleic acids from a sample, comprising: a channel; a constriction region located along a length of the channel; a first valve coupled to the channel; a second valve coupled to the channel; and a collection region fluidically coupled to the constriction region.

60. The device of claim 59, further comprising: an inlet port fluidically coupled to the channel; an outlet port fluidically coupled to the channel; a fluid flow generator configured to generate a fluidic flow of the sample at a flow rate between the inlet port and the outlet port via the channel, wherein the sample comprises the nucleic acids; and an electric field generator configured to generate an electric field that causes an electrophoretic flow that is opposite to the fluidic flow, wherein a combination of the fluidic flow and the electrophoretic flow generates an electrohydrodynamic force to concentrate the nucleic acids in a region comprising the constriction region thereby yielding concentrated nucleic acids.

61. The device of claim 60, wherein the channel comprises a microchannel of a microfluidic device.

62. The device of claim 61, wherein the microfluidic device comprises two or more instances of the microchannel each comprising a constriction region configured to extract nucleic acids from a sample.

63. The device of claim 61, wherein the microfluidic device is fabricated from one or more combinations of polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polyethylene, or glass.

64. The device of claim 60, wherein a length of the channel between the inlet port and the constriction region is from about 0.001 pm to about 100 mm.

65. The device of claim 60, wherein a length of the channel between the inlet port and the constriction region is about 10 mm.

66. The device of claim 60, wherein a length of the channel between an end of the constriction region to an opposite end of the constriction region is from about 0.001 pm to about 100 mm.

67. The device of claim 60, wherein a length of the channel between an end of the constriction region to an opposite end of the constriction region is about 10 mm.

68. The device of claim 60, wherein a length of the channel between an end of the constriction region to the outlet port is from about 0.001 pm to about 100 mm.

69. The device of claim 60, wherein a length of the channel between an end of the constriction region to the outlet port is about 20 mm.

70. The device of claim 60, wherein a width of the channel at regions other than the constriction region is from about 0.001 pm to about 10 mm.

71. The device of claim 60, wherein a width of the channel at regions other than the constriction region is about 1 mm.

72. The device of claim 60, wherein a width of the constriction region is from about 0.001 pm to about 100 mm.

73. The device of claim 60, wherein a width of the constriction region is about 1 mm.

74. The device of claim 60, wherein the constriction region has a height ranging from about 0.001 nm to about 100 mm.

75. The device of claim 60, wherein the constriction region has a height ranging from about 0.01 nm to about 10 mm.

76. The device of claim 60, wherein the constriction region has a height ranging from about 0.1 nm to about 1 mm.

77. The device of claim 57, wherein the constriction region has a height greater than 100 mm.

78. The device of claim 60, wherein the constriction region has a height of about 225 pm.

79. The device of claim 60, wherein the constriction region of the channel has a cross-sectional width that is a factor of at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 1 lx, 12x, 13x, 14x,15x, 16x, 17x, 18x, 19x, or 20x wider than the channel.

80. The device of claim 60, wherein the constriction region comprises one or more comers each with a fillet radius ranging from about 0 pm to about 200 pm.

81. The device of claim 60, wherein the constriction region comprises one or more comers each with a fillet radius ranging from about 10 pm to about 100 pm.

82. The device of claim 60, wherein the constriction region comprises one or more comers each with a fillet radius ranging from about 20 pm to about 50 pm.

83. The device of claim 60, wherein an entrance zone to the region comprising the constriction region has a shape selected from square, rectangular, circular, hyperbolic, or semi-circular.

84. The device of claim 60, wherein the fluidic flow of the sample through the channel is at a volumetric flow rate of from about 0.001 pL / min to about 1000 pL / min.

85. The device of claim 60, wherein the fluidic flow of the sample through the channel is at a flow speed of from about 1 pm / s to about 500 mm / s.

86. The device of claim 60, wherein the electric field has a field strength of from about 0.001 mV / cm to about 10 kV / cm.

87. The device of claim 60, wherein: a) the electric field has a field strength; b) the fluidic flow has a volumetric flow rate; and c) the field strength and volumetric flow rate establish a field strength: volumetric flow rate ratio of about 4 V / cm: 1 pL / min.

88. The device of claim 57, wherein the electrohydrodynamic force causes the nucleic acids to substantially concentrate in an outer region of the region comprising the constriction region.

89. The device of claim 60, wherein the electrohydrodynamic force causes the nucleic acids to substantially concentrate at a top portion, a bottom portion, a left side portion, a right side portion, or any combination therefore of the region comprising the constriction.

90. The device of claim 60, wherein the electric field generator generates a first voltage at an electrode at a first position and a second voltage at an electrode at a second position, wherein the first position and the second position are located on opposite sides of the constriction region.

91. The device of claim 90, wherein the electric field generated by the first and second voltages is substantially uniform along the length of the channel.

92. The device of claim 90, wherein the first and the second voltages are set to establish the electrophoretic flow that is opposite to the fluidic flow.

93. The device of claim 90, wherein the first and second voltages are generated outside of the channel and electrically coupled to the channel.

94. The device of claim 90, wherein the first and second voltages are generated proximate to the channel and electrically coupled to the channel.

95. The device of claim 90, wherein the first and second voltages are generated at microelectrodes integrated into the channel and electrically coupled to the channel.

96. The device of claim 60, further comprising a third valve and a fourth valve, wherein the third valve and the fourth valve are configured to stop the fluidic flow of the sample through the channel.

97. The device of claim 60, wherein the first valve and the second valve are configured to isolate the region comprising the constriction region from other regions of the channel thereby creating an isolated constriction region.

98. The device of claim 60, wherein the first and second valves comprise a pneumatic valve.

99. The device of claim 60, wherein the first and second valves are arranged to isolate the region comprising the constriction region.

100. The device of claim 60, wherein the first and second valves are arranged proximate to the region comprising the constriction region.

101. The device of claim 60, wherein the first and second valves isolate the region comprising the constriction region and wherein the region contains a volume of about 10 pL.

102. The device of claim 60, further comprising a third valve concentric with the constriction region and configured to extract the concentrated nucleic acids from a collection region.

103. The device of claim 60, wherein the collection region is coupled to a collection device.

104. The device of claim 103, wherein the collection device is configured to extract concentrated nucleic acids from the collection region.

105. The device of claim 59, wherein the sample comprises the nucleic acids.

106. The device of claim 59, wherein the sample comprises proteins.

107. The device of claim 59, wherein the sample comprises a cell lysate sample.

108. The device of claim 59, wherein the sample comprises a biological fluid.

109. The device of claim 108, wherein the biological fluid comprises cellular nucleic acids.

110. The device of claim 108, wherein the biological fluid comprises a whole blood sample, a plasma sample, a serum sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, or a lung exudate sample.

111. The device of claim 59, wherein the sample comprises a buffer.

112. The device of claim 111, wherein the buffer has an ionic concentration ranging from about 0 mM to about 100 mM.

113. The device of claim 111, wherein the buffer has an ionic concentration of greater than about 100 mM.

114. The device of claim 111, wherein the buffer has a pH ranging from about 6.5 to about 8.5.

115. The device of claim 59, wherein the nucleic acids comprise deoxyribonucleic acids, ribonucleic acids, or combinations thereof.

116. The device of claim 60, further comprising an element configured to heat the sample, wherein the element is integrated into the device or is coupled to the device.

117. The device of claim 60, further comprising an element configured to mix the sample, wherein the element is integrated into the device or is coupled to the device.

118. The device of claim 60, further comprising an element configured to purify the sample, wherein the element is integrated into the device or is coupled to the device.

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