CN117715701A

CN117715701A - Method and system for droplet manipulation

Info

Publication number: CN117715701A
Application number: CN202280032444.6A
Authority: CN
Inventors: 威廉·凯·兰福德; 阿基姆·伦霍夫; 德文·莱尔·詹金斯; 伊沙安·戈文达拉詹; 阿卜杜勒·马吉德·穆罕默德; 拉斐尔·梅兰德; 利亚姆·马斯特斯; 尤德彦·乌玛帕西
Original assignee: Volta Laboratories Inc
Current assignee: Volta Laboratories Inc
Priority date: 2021-03-02
Filing date: 2022-03-02
Publication date: 2024-03-15

Abstract

Described herein are systems and methods for performing various biological assays on an array using electrowetting on dielectric (EWOD). The systems and methods may process a biological sample or multiple biological samples using at least one droplet. The droplet or droplets can be manipulated using the systems and methods described herein. Improvements to arrays are also described herein to facilitate performing biological assays on the arrays.

Description

Method and system for droplet manipulation

Cross reference

The present application claims the benefit of U.S. provisional application number 63/155,692 filed on 3/2/2021, U.S. provisional application number 63/250,101 filed on 9/29/2021, U.S. provisional application number 63/255,721 filed on 10/14/2021, and U.S. provisional application number 63/287,412 filed on 8/2021, the entire contents of which are incorporated herein by reference.

Background

Biological samples may be processed for various applications. For example, deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules can be processed (e.g., sequenced) to identify genetic variants, which can be used to identify diseases such as cancer. These biological samples can be processed in a divided state such as a droplet. The sequence of DNA or RNA can be determined by sequence identification, such as nucleic acid sequencing.

Droplets containing biological samples can be manipulated using electrowetting, which can use an electric field from an electrode to move the droplet adjacent to a surface.

Disclosure of Invention

Some aspects of the present disclosure provide methods for circularizing a nucleic acid sample, comprising: providing a droplet adjacent to an electrowetting array, wherein the sample droplet comprises the nucleic acid sample, and treating the droplet using the electrowetting array to circularize the nucleic acid sample. In some embodiments, the electrowetting array comprises a dielectric matrix. In some embodiments, the electrowetting array further comprises one or more reagent droplets. In some embodiments, the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample. In some embodiments, the method further comprises: combining the sample droplet with one or more reagent droplets; separating the sample droplets from the one or more reagents; and combining the one or more reagent droplets with a second droplet. In some embodiments, the droplet comprises one or more reagents for circularizing the nucleic acid sample. In some embodiments, (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets. In some embodiments, the electrowetting array comprises one or more electrodes below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the electrowetting array. In some embodiments, the method further comprises performing a sequencing reaction on the nucleic acid sample using a single polymerase. In some embodiments, the method further comprises generating a sequencing read that is at least 70 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is at least 80 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is about 200 kilobases (kb) in length. In some embodiments, at least 100 (Gb) of sequencing data is generated. In some embodiments, at least 500Gb of sequencing data is generated. In some embodiments, at least 512Gb of sequencing data is generated. In some embodiments, at least 10Gb of data is generated. In some embodiments, at least 30Gb of data is generated. In some embodiments, the sequencing reaction comprises repeated cycle sequencing. In some embodiments, one or more sub-reads of the sequencing reads are generated. In some embodiments, a consensus sequence is generated from the sub-reads of a sequencing read. In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84. In some embodiments, the method further comprises generating a circularized nucleic acid sample. In some embodiments, the circularized nucleic acid sample comprises a target sequence. In some embodiments, the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence. In some embodiments, at least 80% of the plurality of sequences comprise the target sequence. In some embodiments, the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

Another aspect of the disclosure is a method of sequencing a nucleic acid sample comprising (a) providing a droplet adjacent to an electrowetting array, the droplet comprising the nucleic acid sample, (b) treating the droplet with the electrowetting array to circularize the nucleic acid sample, and (c) performing a sequencing reaction on the circularized nucleic acid sample using a single polymerase. A method for sequencing a circular nucleic acid sample comprising performing a sequencing reaction on the nucleic acid sample using a single polymerase to produce a sequencing read of at least 70 kilobases in length. In some embodiments, the method further comprises using a waveguide to detect bases incorporated into the nucleic acid sample during the sequencing reaction. In some embodiments, the electrowetting array comprises a dielectric matrix. In some embodiments, the electrowetting array further comprises one or more reagent droplets. In some embodiments, the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample. In some embodiments, the method further comprises combining the sample droplet with one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. In some embodiments, the droplet comprises one or more reagents for circularizing the nucleic acid sample. In some embodiments, (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets. In some embodiments, the electrowetting array comprises one or more electrodes below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the electrowetting array. In some embodiments, the method further comprises performing a sequencing reaction on the nucleic acid sample using a single polymerase. In some embodiments, the method further comprises generating a sequencing read that is at least 70 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is at least 80 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is about 200 kilobases (kb) in length. In some embodiments, at least 100 (Gb) of sequencing data is generated. In some embodiments, at least 500Gb of sequencing data is generated. In some embodiments, at least 512Gb of sequencing data is generated. In some embodiments, at least 10Gb of data is generated. In some embodiments, at least 30Gb of data is generated. In some embodiments, the sequencing reaction comprises repeated cycle sequencing. In some embodiments, one or more sub-reads of the sequencing reads are generated. In some embodiments, a consensus sequence is generated from the sub-reads of a sequencing read. In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84. In some embodiments, the method further comprises generating a circularized nucleic acid sample. In some embodiments, the circularized nucleic acid sample comprises a target sequence. In some embodiments, the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence. In some embodiments, at least 80% of the plurality of sequences comprise the target sequence. In some embodiments, the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

Another aspect of the present disclosure is a method of producing a circularized nucleic acid sample having a longer insert size comprising (a) providing a droplet comprising a nucleic acid sample adjacent to an electrowetting array, (b) treating the droplet with the electrowetting array to circularize the nucleic acid sample, and (c) performing a sequencing reaction on the circularized nucleic acid sample using a single polymerase. In some embodiments, the electrowetting array comprises a dielectric matrix. In some embodiments, the electrowetting array further comprises one or more reagent droplets. In some embodiments, the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample. In some embodiments, the method further comprises combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. In some embodiments, the droplet comprises one or more reagents for circularizing the nucleic acid sample. In some embodiments, (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets. In some embodiments, the electrowetting array comprises one or more electrodes below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the electrowetting array. In some embodiments, the method further comprises performing a sequencing reaction on the nucleic acid sample using a single polymerase. In some embodiments, the method further comprises generating a sequencing read that is at least 70 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is at least 80 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is about 200 kilobases (kb) in length. In some embodiments, at least 100 (Gb) of sequencing data is generated. In some embodiments, at least 500Gb of sequencing data is generated. In some embodiments, at least 512Gb of sequencing data is generated. In some embodiments, at least 10Gb of data is generated. In some embodiments, at least 30Gb of data is generated. In some embodiments, the sequencing reaction comprises repeated cycle sequencing. In some embodiments, one or more sub-reads of the sequencing reads are generated. In some embodiments, a consensus sequence is generated from the sub-reads of a sequencing read. In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84. In some embodiments, the method further comprises generating a circularized nucleic acid sample. In some embodiments, the circularized nucleic acid sample comprises a target sequence. In some embodiments, the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence. In some embodiments, at least 80% of the plurality of sequences comprise the target sequence. In some embodiments, the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

Another aspect of the disclosure is a method for generating a sequencing library, comprising (a) providing a nucleic acid sample comprising a plurality of nucleic acid molecules, the nucleic acid molecules comprising a plurality of sequences, and (b) generating the sequencing library using the nucleic acid sample, wherein the sequencing library comprises at least 80% of the plurality of sequences of its complement; (c) Processing the droplets using the electrowetting array to circularize the nucleic acid sample; (d) Separating the droplets from the one or more reagent droplets; and; (e) Combining the one or more reagent droplets with the sample droplet to generate a circularized nucleic acid sample. In some embodiments, the electrowetting array comprises a dielectric matrix. In some embodiments, the electrowetting array further comprises one or more reagent droplets. In some embodiments, the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample. In some embodiments, the method further comprises combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. In some embodiments, the droplet comprises one or more reagents for circularizing the nucleic acid sample. In some embodiments, (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets. In some embodiments, the electrowetting array comprises one or more electrodes below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the electrowetting array. In some embodiments, the method further comprises performing a sequencing reaction on the nucleic acid sample using a single polymerase. In some embodiments, the method further comprises generating a sequencing read that is at least 70 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is at least 80 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is about 200 kilobases (kb) in length. In some embodiments, at least 100 (Gb) of sequencing data is generated. In some embodiments, at least 500Gb of sequencing data is generated. In some embodiments, at least 512Gb of sequencing data is generated. In some embodiments, at least 10Gb of data is generated. In some embodiments, at least 30Gb of data is generated. In some embodiments, the sequencing reaction comprises repeated cycle sequencing. In some embodiments, one or more sub-reads of the sequencing reads are generated. In some embodiments, a consensus sequence is generated from the sub-reads of a sequencing read. In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84. In some embodiments, the method further comprises generating a circularized nucleic acid sample. In some embodiments, the circularized nucleic acid sample comprises a target sequence. In some embodiments, the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence. In some embodiments, at least 80% of the plurality of sequences comprise the target sequence. In some embodiments, the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

Another aspect of the disclosure is a method for circularizing a nucleic acid sample, comprising: providing a droplet adjacent to an electrowetting array, wherein the droplet comprises the nucleic acid sample; combining the droplets with one or more reagent droplets; processing the droplets using the electrowetting array to circularize the nucleic acid sample; separating the droplets from the one or more reagent droplets, and combining the one or more reagent droplets with the sample droplet to generate a circularized nucleic acid sample. In some embodiments, the electrowetting array comprises a dielectric matrix. In some embodiments, the electrowetting array further comprises one or more reagent droplets. In some embodiments, the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample. In some embodiments, the method further comprises: combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. In some embodiments, the droplet comprises one or more reagents for circularizing the nucleic acid sample. In some embodiments, (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets. In some embodiments, the electrowetting array comprises one or more electrodes below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the one or more reagent droplets, the sample droplet, or both. In some embodiments, the one or more droplet operations include applying vibration to the electrowetting array. In some embodiments, the method further comprises performing a sequencing reaction on the nucleic acid sample using a single polymerase. In some embodiments, the method further comprises generating a sequencing read that is at least 70 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is at least 80 kilobases (kb) in length. In some embodiments, the method further comprises generating a sequencing read that is about 200 kilobases (kb) in length. In some embodiments, at least 100 (Gb) of sequencing data is generated. In some embodiments, at least 500Gb of sequencing data is generated. In some embodiments, at least 512Gb of sequencing data is generated. In some embodiments, at least 10Gb of data is generated. In some embodiments, at least 30Gb of data is generated. In some embodiments, the sequencing reaction comprises repeated cycle sequencing. In some embodiments, one or more sub-reads of the sequencing reads are generated. In some embodiments, a consensus sequence is generated from the sub-reads of a sequencing read. In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84. In some embodiments, the method further comprises generating a circularized nucleic acid sample. In some embodiments, the circularized nucleic acid sample comprises a target sequence. In some embodiments, the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence. In some embodiments, at least 80% of the plurality of sequences comprise the target sequence. In some embodiments, the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

Another aspect of the present disclosure is a method of producing a biopolymer, comprising: providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent; moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet and (ii) form a combined droplet comprising the first reagent and the second reagent; and forming at least a portion of the biopolymer using at least (i) the first reagent and (ii) the second reagent in the combined droplet, wherein (b) - (c) are performed in a period of 10 minutes or less. In some embodiments, the biopolymer is a polynucleotide. In some embodiments, the biopolymer is a polypeptide. In some embodiments, wherein the polynucleotide comprises about 10 to about 250 bases. In some embodiments, wherein the polynucleotide comprises about 260 to about 1kb. In some embodiments, the polynucleotide comprises about 1kb to about 10,000kb. In some embodiments, vibration is applied to the synthetic droplets during (b), (c), or both. In some embodiments, the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets. In some embodiments, vibration is applied to the one or more cleaning steps. In some embodiments, the surface is a dielectric. In some embodiments, the surface includes a dielectric layer disposed over the one or more electrodes. In some embodiments, the surface is a surface of a polymer film. In some embodiments, the surface comprises one or more oligonucleotides bound to the surface. In some embodiments, the surface is a surface of a lubricating liquid layer. In some embodiments, the plurality of droplets includes a third droplet comprising a third reagent. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof, comprises one or more functionalized beads. In some embodiments, the functionalized bead comprises one or more oligonucleotides immobilized thereto. In some embodiments, vibration is applied to the first droplet, the second droplet, the third droplet, the wash droplet, or a mixture thereof. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer. In some embodiments, the biomonomer is an amino acid. In some embodiments, the biomonomer is a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises one or more functionalized discs. In some embodiments, the functionalized disc comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof, comprises an enzyme that mediates synthesis or polymerization. In some embodiments, the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 20 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 15 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 10 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 1 minute or less. In some embodiments, the combined droplets are temperature controlled. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is exposed to light. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps). In some embodiments, the deoxynucleoside triphosphates can have a protecting group. In some embodiments, the protecting group may be removed in the reaction. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side. In some embodiments, the first, second, third, or combined droplet has a volume between 1 nanoliter (1 nL) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 200 microliters (200 μl). In some embodiments, the method further comprises attaching the biopolymer to a second biopolymer. In some embodiments, the second biopolymer is produced using any of the methods disclosed herein.

Another aspect of the present disclosure provides a method of producing a biopolymer, comprising: providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent; moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet, and (ii) form a combined droplet comprising the first reagent and the second reagent; and forming at least a portion of the biopolymer using at least (i) the first reagent and (ii) the second reagent in the combined droplet, wherein vibration is applied to (b), (c), or both. In some embodiments, the biopolymer is a polynucleotide. In some embodiments, the biopolymer is a polypeptide. In some embodiments, the polynucleotide comprises 2 to 10,000,000 nucleic acid molecules. In some embodiments, the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets. In some embodiments, vibration is applied to the one or more cleaning steps. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 30 minutes or less. In some embodiments, the surface is a dielectric. In some embodiments, the surface includes a dielectric layer disposed over the one or more electrodes. In some embodiments, the surface is a surface of a polymer film. In some embodiments, the surface comprises one or more oligonucleotides bound to the surface. In some embodiments, the surface is a surface of a lubricating liquid layer. In some embodiments, the plurality of droplets includes a third droplet comprising a third reagent. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof, comprises one or more functionalized beads. In some embodiments, the functionalized bead comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer. In some embodiments, the biomonomer is an amino acid. In some embodiments, the biomonomer is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the first agent comprises one or more functionalized discs. In some embodiments, the functionalized disc comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first droplet, the second droplet, the third droplet, or a combination thereof comprises an enzyme that mediates synthesis or polymerization. In some embodiments, the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 20 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 15 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 10 minutes or less. In some embodiments, the combined droplets are heated. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is exposed to light. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps). In some embodiments, the deoxynucleoside triphosphates can have a protecting group. In some embodiments, the protecting group may be removed in the reaction. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side. In some embodiments, the first, second, third, or combined droplet has a volume between 1 nanoliter (1 nl) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume of between 1 microliter (1 ul) and 500 microliters (500 ul). In some embodiments, the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 microliter (1 ul) and 200 microliters (200 μl).

Another aspect of the disclosure includes a method for processing a nucleic acid sample, comprising: providing a biological sample adjacent to an electrowetting array, wherein the sample droplet comprises the nucleic acid sample; and extracting the nucleic acid sample from the biological sample adjacent to the electrowetting array, wherein the nucleic acid sample comprises a sequencing read that is at least about 70 kilobases (kb) in length. In some embodiments, the length is at least about 80 kilobases (kb). In some embodiments, the length is at least about 200 kilobases (kb). In some embodiments, the sequencing reads have an a260/a280 ratio of less than about 1.84.

Another aspect of the present disclosure provides a system for inducing droplet motion, comprising: (a) a surface configured to support the droplet, the droplet comprising at least one bead formed of a material configured to couple to a magnetic field, (b) an actuator coupled to a magnet, wherein the magnet is configured to provide the magnetic field, and wherein the actuator is configured to translate the magnetic field along a plane parallel to the surface, and (c) a controller operably coupled to the actuator, wherein the controller is configured to instruct the actuator to translate the magnetic field along the plane such that the droplet performs a motion along the surface as the magnetic field translates along the plane. In some embodiments, the actuator is a switch. In some embodiments, the actuator includes a motor coupled to the magnet, wherein the motor is configured to translate the magnet in a direction parallel to the surface. In some embodiments, the system further comprises electrodes configured to provide an electric field to the surface, wherein the electric field and the magnetic field are sufficient to cause the movement of the droplet. In some embodiments, the actuator is configured to move the magnet to translate it along at least two axes parallel to the plane. In some embodiments, the magnet comprises a permanent magnet. In some embodiments, the magnet comprises at least one electromagnet. In some embodiments, the actuator comprises a pivot, wherein the pivot is coupled to the surface. In some embodiments, the surface includes a dielectric disposed over the one or more electrodes. In some embodiments, the one or more magnets are disposed below the surface. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises a liquid having an affinity for the surface.

Another aspect of the present disclosure provides a system for processing a sample, comprising: (a) a plurality of electrodes, (b) a dielectric layer disposed over the plurality of electrodes, wherein the dielectric layer comprises a surface configured to support a droplet comprising the sample, and (c) a liquid disposed in a gap adjacent to the plurality of electrodes and the dielectric layer. In some embodiments, the liquid creates an adhesion between the plurality of electrodes and the dielectric layer. In some embodiments, the liquid comprises a dielectric material. In some embodiments, the liquid prevents or reduces the conductivity of air disposed in the gap. In some embodiments, the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm (paramilm), polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the dielectric layer is removable. In some embodiments, the attachment is sufficient to secure the liquid to the surface, and wherein the liquid is resistant to gravity. In some embodiments, the liquid is selected to preferentially wet the surface to facilitate movement of the liquid droplet on the surface.

Drawings

The novel features of the 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 exemplary embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "figures") of which:

fig. 1 shows a side cross-sectional view of a printed circuit board with various steps of a dielectric coating and various steps of a planarization process.

FIG. 2 shows a sketch of a computer system for the arrays described herein.

Fig. 3A-3B illustrate example systems and methods for configuring reference electrodes on an electrode array. Fig. 3A shows a liquid coating used as a reference electrode. Fig. 3B shows electrically conductive ionized particles.

FIG. 4 depicts size distribution data for DNA isolated using the systems and methods described herein.

Fig. 5A-5C depict configurations of synthesizing and assembling biopolymers (e.g., DNA) using the systems and methods described herein. Fig. 5A and 5B show an example workflow for DNA synthesis. FIG. 5C shows a schematic of a single reaction site at which stepwise addition of nucleotides is performed to synthesize long molecules of DNA.

Figure 6 shows library size distributions for on-chip and off-chip experiments prepared using NGS libraries of the systems and methods described herein.

Figure 7 depicts the quality of sequencing libraries for on-chip and off-chip experiments prepared using NGS libraries of the systems and methods described herein.

Figure 8 depicts the repeat levels of sequencing libraries for on-chip and off-chip experiments prepared using NGS libraries of the systems and methods described herein.

Figures 9A-9B depict aptamer contamination levels for NGS library preparation experiments.

Figure 10 depicts the horizontal coverage of NGS library preparation experiments throughout the human genome using the systems and methods described herein.

FIG. 11 depicts Single Nucleotide Polymorphism (SNP) sensitivity of an NGS library preparation experiment using the systems and methods described herein.

Fig. 12 depicts an illustrative NGS workflow example using the systems and methods described herein. Examples of workflows include manipulating (e.g., lysing cells, digesting proteins, and DNA clearing) biological samples on an array as described herein.

Fig. 13A-13B depict arrays according to some embodiments described herein.

Fig. 14A-14B depict arrays according to some embodiments described herein.

Fig. 15 depicts a circuit diagram of a system without a dedicated reference electrode as described herein.

FIGS. 16A-16B illustrate one application of vibration assisted electrowetting on a dielectric for extracting DNA using magnetic beads.

Fig. 17A-17B show that the response of a high contact angle droplet (fig. 17A) to vibration has a greater tendency than a droplet with a low contact angle (fig. 17B).

Fig. 18A-18B depict embodiments of an electromechanical actuator.

Fig. 19A-19B depict other embodiments of electromechanical actuators.

Fig. 20 shows an embodiment that efficiently couples the driving force of an electromechanical actuator into the droplet vibration and ultimately achieves efficient mixing.

Fig. 21 illustrates an embodiment of adjusting the natural frequency of the vibration assisted EWOD system to be low relative to the desired frequency range.

Fig. 22 illustrates an embodiment of the present disclosure that includes evaporation control of one or more relevant droplets on an array described herein with oil.

Fig. 23 illustrates one embodiment of the present disclosure that includes a nucleic acid sequencing assay (e.g., nanopore sequencing) integrated into an array described herein.

Fig. 24 shows the results of extracting High Molecular Weight (HMW) DNA from GM12878 cells using the methods and apparatus of the present disclosure.

Figures 25A-25C show the results of extracting High Molecular Weight (HMW) DNA from whole human blood samples using the methods and apparatus of the present disclosure.

26A-26B illustrate improved gDNA extraction results using the methods and apparatus of the present disclosure as compared to manual sample and/or reagent processing.

FIG. 27 shows improved gDNA extraction results using the methods and apparatus of the present disclosure, as compared to manual sample and/or reagent processing.

FIG. 28 shows improved sequencing results on a MinION sequencing system for gDNA extracted using the methods and apparatus of the present disclosure, as compared to manual sample and/or reagent processing.

FIG. 29 shows improved sequencing data generated on a MinION sequencing system using gDNA extracted by the methods and apparatus of the present disclosure.

FIG. 30 shows improved sequencing data generated on a MinION sequencing system using gDNA extracted by the methods and apparatus of the present disclosure.

FIGS. 31A-31B show the distribution of library fragment sizes for one GM12878 and one whole blood sample extracted using the methods and apparatus of the present disclosure.

FIGS. 32A-32B show a distribution of read lengths on a Minion sequencing system for gDNA extracted using the methods and apparatus of the present disclosure.

FIGS. 33A-33E show sequencing results on a Pacific Biosciences of California HiFi sequencing system of gDNA extracted using the methods and apparatus of the present disclosure; including read length (fig. 33A), sub-read length (fig. 33B), read quality distribution (fig. 33C), hiFi read length distribution (fig. 33D), and a model of expected accuracy versus read length (fig. 33E).

Fig. 34 shows an increase in average concentration/purity of DNA extracted using the methods and apparatus of the present disclosure.

35A-35B illustrate experimental enhancements and workflow robustness enhancements of the methods and apparatus as the methods and apparatus of the present disclosure are further utilized.

36A-36B illustrate embodiments of the systems and devices described herein that include a movable magnet for inducing movement of magnetically responsive material contained in droplets on the arrays/substrates described herein.

Detailed Description

While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the disclosure. It should be understood that the embodiments of the present disclosure described herein may employ various alternatives.

The term "at least," "greater than," or "greater than or equal to" when used in reference to a first value of a sequence of two or more values applies to each value of the sequence of values. For example, 1 or more, 2 or 3 or more is 1 or more, 2 or more or 3 or more.

The term "no greater than", "less than" or "less than or equal to" when used in reference to a first value of a sequence of two or more values applies to each value of the sequence of values. For example, 3 or less, 2 or 1 corresponds to 3 or less, 2 or less or 1 or less.

The term "slip angle" as used herein generally refers to the angle a droplet of a given size forms with the horizontal as it begins to move under the force of gravity. For example, a 5 microliter (μl) hair drop is held at 4 °, but a surface that allows it to slide at 5 ° may have a 5 μl sliding angle of 5 °. For various applications, a sliding angle of 5 μl of less than or equal to 70 °, 60 °, 50 °, 40 °, 30 °, 25 °, 20 °, 15 °, 10 °, 5 °, 3 °, 2 °, 1 °, or less may be used. The smaller the slip angle, the smoother the surface and generally the lower the voltage required to move the droplet across the surface.

The term "contact angle hysteresis" as used herein generally refers to the difference observed between advancing and receding contact angles. For example, at a surface with lower surface adhesion, the contact angle of the leading edge with the surface and the contact angle of the trailing edge with the surface may be nearly the same as the droplet moves across the surface. However, at a surface with higher adhesion, the difference between the front contact angle and the back contact angle may become larger. Low surface roughness, high surface hydrophobicity and low surface energy will result in less difference in the angle. Contact angle hysteresis (i.e., the difference between the anterior contact angle and the posterior contact angle) of less than or equal to 70 °, 60 °, 50 °, 40 °, 30 °, 25 °, 20 °, 15 °, 10 °, 7 °, 5 °, 3 °, 2 ° or less may be used.

The term "droplet" as used herein generally refers to a discrete or finite volume of fluid (e.g., liquid). Droplets may be generated from one phase separated from another phase by an interface. The droplets may be a first phase separated from another phase. The droplets may comprise a single phase or multiple phases (e.g., an aqueous phase containing a polymer). The droplets may be a liquid phase disposed adjacent to the surface and in contact with another phase (e.g., a gas phase such as air).

The term "biological sample" as used herein generally refers to biological material. These biological materials may exhibit biological activity or be biologically active. These biological materials may be or may include deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, polypeptides (e.g., proteins), or any combination thereof. The biological sample (or specimen) may be a tissue sample, such as a biopsy, a core biopsy, a needle aspirate, or a fine needle aspirate. The sample may be a fluid sample, such as a blood sample, a urine sample, a stool sample, or a saliva sample. The sample may be a skin sample. The sample may be an oral swab. The sample may be a plasma or serum sample. The sample may be a sample of plant origin, a water sample or a soil sample. The sample may be from an alien. The alien sample may contain biological material. The sample may be a cell-free (or cell-free) sample. The cell-free sample may comprise extracellular polynucleotides. The extracellular polynucleotides may be isolated from a body sample, which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The sample may comprise a eukaryotic cell or a plurality of eukaryotic cells. The sample may comprise a prokaryotic cell or a plurality of prokaryotic cells. The sample may comprise a virus. The sample may comprise a compound derived from an organism. The sample may be from a plant. The sample may be from an animal. The sample may be from an animal suspected of having or carrying the disease. The sample may be from a mammal.

The term "% glycerol" as used herein generally refers to the viscosity of a solution as compared to glycerol in an aqueous solution, wherein the amount of glycerol in water (by volume) is determined by a percentage value. For example, a solution having a viscosity of about "30% glycerol" as described herein means that the viscosity of the solution corresponds to an aqueous glycerol solution containing about 30% glycerol.

The term "subject" as used herein generally refers to an animal, such as a mammal (e.g., a human) or bird (e.g., a bird), or other organism, such as a plant. The subject may be a vertebrate, mammal, rodent (e.g., mouse), primate, simian, or human. Animals may include, but are not limited to, farm animals, sports animals, and pets. The subject may be a healthy or asymptomatic individual, an individual who has or is suspected of having a disease (e.g., cancer) or is susceptible to the disease, an individual in need of treatment or suspected of being in need of treatment, or any combination thereof. The object may be a patient.

The term "electromechanical actuator" as used herein generally refers to a non-human structure that may be used to apply vibration and/or acoustic forces to an array as described herein. As non-limiting examples, electromechanical actuators include oscillating mechanisms or cantilevers, motor-driven links, and/or rotating masses. In some embodiments, the electromechanical actuators described herein are flexible structures that include various flexible elements (e.g., linear flexures) or have conventional bearings.

The term "coefficient of variation" as used herein generally refers to repeatability and accuracy. This can be expressed by equation 1, where s is the standard deviation of the responsivity of the different materials and x is the average responsivity of all materials.

The term "crosstalk" as used herein generally refers to contamination of droplets. Crosstalk may refer to the percentage of a droplet, biological sample, or a combination thereof that is collected from another droplet. P (P) ₁ Represents the relevant substances in the liquid drop, and P ₂ The crosstalk, which is the total material from other droplets present in the relevant droplet, can be expressed by equation 2.

Electrowetting device and system

Electrowetting devices may be used to move individual droplets of water (or other aqueous, polar or conductive solutions) from one place to another. The surface tension and wetting properties of water can be changed by the electric field strength utilizing the electrowetting effect. The electrowetting effect may result from a change in the solid-liquid contact angle caused by an applied potential difference between the solid and the liquid. The difference in wetting surface tension, which can vary across the width of the droplet, and the corresponding variation in contact angle can provide the motive force to move the droplet without moving parts or physical contact. The electrowetting device may comprise an electrode grid with a dielectric layer of suitable electrical and surface priority overlying the electrodes, all laid on a rigid insulating substrate. Other examples of electrowetting devices may be found in WO2021041709, the entire content of which is incorporated herein by reference.

The surface of the electrode grid may be made so that it has low adhesion to water. Thus, the water droplets can be moved along the surface by a minute force generated by an electric field and a surface tension gradient across the width of the droplets. Surfaces with low adhesion can reduce the trajectories left by the droplets. Smaller trajectories can reduce droplet cross-contamination and can reduce sample loss during droplet movement. The low adhesion to the surface also allows the drive voltage for the droplet motion to be reduced and the reproducibility of the droplet motion to be achieved. For example, using a contact angle goniometer or Charge Coupled Device (CCD) camera, there are several methods that can measure low adhesion between the surface and the droplet, including slip angle and contact angle hysteresis.

There are several ways to achieve low surface adhesion; for example, mechanical polishing, chemical etching, or a combination thereof is used to achieve a smoothness on the order of a few nanometers, a coating is applied to compensate for surface irregularities, a liquid is added to compensate for surface irregularities, and the surface is chemically modified to produce desired surface characteristics (hydrophobicity, hydrophilicity, bioadhesion resistance, variation in electric field strength, etc.).

Liquid-to-liquid electrowetting for electrowetting (LLEW)

An electrowetting mechanism called "liquid-to-liquid electrowetting" (LLEW) exploits the electrowetting phenomenon that occurs at the liquid-gas interface. Droplets adhering to the surface of the low surface energy liquid (e.g., oil) layer and substantially surrounded by gas (e.g., air, nitrogen, argon, etc.) form a liquid-gas interface at the contact line. The oil may be stabilized at a designated location on the solid substrate by the textured surface of the solid substrate, and the conductive layer of the metal electrode may be embedded in the body of the solid. In some embodiments, when an electrical potential is applied across the height of the droplet, the liquid-gas interface may cause the droplet to wet the oil and spread across the surface while still adhering to the oil.

In some embodiments, liquid-to-liquid electrowetting techniques may be used to manipulate droplets that may contain biological and chemical samples. In some embodiments, the droplet may move from left to right and may be attracted to the leftmost electrode of the three electrodes by a positive voltage of the leftmost electrode, thereby increasing the electric field at the liquid-liquid surface and promoting wetting. In some embodiments, the voltage is withdrawn from the leftmost electrode and applied to the center electrode. In some embodiments, the droplet may be attracted to the center location due to the enhanced wettability over the center electrode. In some embodiments, the voltage is withdrawn from the left and center electrodes and applied to the right electrode, and the enhanced wettability over the right electrode attracts the droplet to the right.

In some embodiments, differential wetting can be used to combine two droplets at the LLEW surface over an electrode array. In some embodiments, two droplets are attracted to the leftmost and rightmost electrodes. In some embodiments, the voltages are removed from the left and right electrodes and applied to the center electrode. Two droplets may be attracted to the center from left and right and begin to merge.

In some embodiments, such microfluidic selective wetting devices are capable of performing microfluidic droplet actuation, such as droplet transport, droplet merging, droplet mixing, droplet splitting, droplet dispensing, droplet shape change, or combinations thereof. The LLEW droplet driver can be used in microfluidic devices for automated biological experiments, such as liquid assays, medical diagnostic devices, and in a variety of lab-on-a-chip applications.

Other examples of LLEW droplet driving can be found in WO2021041709, which is incorporated herein by reference in its entirety.

In some embodiments, a low surface energy liquid (e.g., oil) may be stabilized at a designated location on the solid surface without texturing the solid surface. In these embodiments, the stabilization of the liquid layer depends on the chemical affinity between the surface of the underlying layer and the liquid layer. In some embodiments, the liquid layer is a lubricating film. In some embodiments, the lubricating film is thermodynamically stable, thereby preferentially wetting the surface of the underlying surface. In some embodiments, the underlying surface is a solid matrix. In some embodiments, the solid matrix is a dielectric. In some embodiments, the underlying surface is a film. In some embodiments, the film is a dielectric film. In some embodiments, achieving this stability is important, depending on the affinity of the lubricating liquid for the dielectric surface. In some embodiments, it may be advantageous to use a fluorinated lubricating liquid for the fluorinated surface of the dielectric. Similar chemical structures result in greater affinity and therefore lubricants are more likely to wet the surface in a stable manner. In some embodiments, when using a dielectric body having a hydrocarbon-based surface or a siliconized surface (e.g., silicone resin and untreated polymer plastic), it may be advantageous to use a hydrocarbon-based lubricating liquid (e.g., silicone oil).

One aspect of the present disclosure includes a system for processing a sample, comprising: a plurality of electrodes; a dielectric layer disposed over the plurality of electrodes, wherein the dielectric layer comprises a surface configured to support a droplet comprising a sample; a liquid adjacent to the surface, wherein the liquid has a chemical affinity for the surface sufficient to immobilize the liquid on the surface and the liquid is resistant to gravity. In some embodiments, the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene polyether ether ketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF) perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the dielectric layer is removable.

Electrowetting on dielectric for droplet manipulationWet (EWOD)

In some embodiments, electrowetting on dielectric (EWOD) is a phenomenon in which the wettability of an aqueous, polar or conductive liquid (L) can be modulated by an electric field across the dielectric film between the droplet and the conductive electrode. Increasing or decreasing the charge from the electrode may change the wettability of the insulating dielectric layer, and the change in wettability is reflected in a change in the contact angle of the droplet. The change in contact angle may in turn cause the droplet to change shape, move, break up into smaller droplets, or merge with other droplets. Other examples of EWOD droplet driving can be found in WO2021041709, which is incorporated herein by reference in its entirety.

Attributes of an array

Described herein are arrays and matrices configured to facilitate EWOD to induce droplet actuation. The array may include a plurality of elements, which may include: a plurality of heaters, a plurality of coolers, a plurality of magnetic field generators, a plurality of electroporation units, a plurality of light sources, a plurality of radiation sources, a plurality of nucleic acid sequencers, a plurality of bioprotein channels, a plurality of solid state nanopores, a plurality of protein sequencers, a plurality of acoustic sensors, a plurality of microelectromechanical systems (MEMS) sensors, a plurality of capillaries as liquid dispensers, a plurality of wells for dispensing or transferring liquid using gravity, a plurality of electrodes in a well for dispensing or transferring liquid using an electric field, a plurality of wells for optical detection, a plurality of wells for allowing liquid to interact through a membrane, or any combination thereof. The plurality of elements may include elements less than or equal to about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or less. The plurality of elements may include elements greater than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more.

The heater may have a maximum temperature of less than or equal to about 150 ℃, 125 ℃, 100 ℃, 75 ℃, 50 ℃, 25 ℃ or less. The heater may be thermoelectric, resistive, or heated by a heat transfer medium (e.g., a recirculating hot water loop). The cooler may have a minimum temperature of greater than or equal to about-50 ℃, -25 ℃, -10 ℃, -5 ℃, 0 ℃, 10 ℃ or higher. The cooler may be thermoelectric, evaporative, or cooled by a heat transfer medium (e.g., a water cooler).

The magnetic field generator may be used for magnetic bead based operations or for other operations requiring a magnetic field. The magnetic field generator may be an electromagnet.

The electroporation unit may be two or more electrodes on both sides of the droplet.

The light source may be broadband, monochromatic, or a combination thereof. The light source may be an incandescent light source, a Light Emitting Diode (LED), a laser, or a combination thereof. The light source may emit polarized light, collimated light, or a combination thereof. The plurality of radiation sources may emit ultraviolet light (light having a wavelength of 10nm to 400 nm), X-rays, gamma rays, alpha particles, beta particles, or a combination thereof. The radiation source may be collimated.

Electrowetting matrix

The electrowetting microfluidic device may be formed by forming a smooth (in the sense of a low surface energy) surface directly on the electrode array (120). The electrode array may be composed of conductive plates with dots to drive the droplets. The electrodes in the array may be arranged in any layout, such as a rectangular grid or a collection of discrete paths. The electrode itself may be made of one or more conductive metals (including gold, silver, copper, nickel, aluminum, platinum, titanium), one or more conductive oxides (including indium tin oxide, aluminum doped zinc oxide), one or more conductive organic compounds (including PEDOT and polyacetylene), one or more semiconductors (including silicon dioxide), or any combination thereof. The substrate used to configure the electrode array may be any insulating material of any thickness and any rigidity.

The electrode arrays can be fabricated on standard rigid and flexible printed circuit board substrates. The substrate of the PCB may be FR4 (glass-epoxy), FR2 (glass-epoxy), rogers material (hydrocarbon ceramic) or Insulating Metal Substrate (IMS), polyimide film (exemplary commercial brands include Kapton, pyralux), polyethylene terephthalate (PET), ceramic or other commercially available substrate with a thickness of 1 μm to 10,000 μm. In some embodiments, a thickness from 500 μm to 2000 μm may be used.

The electrode array may also be made of conductive elements, semiconductive elements, or any combination thereof, which may be fabricated using active matrix technology, such as Thin Film Transistor (TFT) technology, and passive matrix technology. The electrode array may also be made of pixel arrays fabricated using conventional CMOS or HV-CMOS fabrication techniques.

The electrode array may also be fabricated by depositing transparent conductive materials such as Indium Tin Oxide (ITO), aluminum doped zinc oxide (AZO), fluorine doped tin oxide (FTO) and the like on glass sheets, polyethylene terephthalate (PET) and any other insulating substrates.

The electrode array may also be fabricated by depositing metal on glass, polyethylene terephthalate (PET), and any other insulating substrate.

In constructing an electrowetting microfluidic device, a multi-layer stack (from 1 layer to 50 layers) may be used to isolate multi-layer electrical interconnect wiring (from 2 layers to 50 layers). One of the outermost layers of the stack may contain an electrode pad for driving the droplet and may contain a reference electrode. The interconnect may connect the electrical pad to a high voltage for driving and for capacitive sensing. The driving voltage may be from 1V to 350V. The driving voltage may be an AC signal or a DC signal.

In a further embodiment, the substrate does not have a dedicated reference electrode. The circuit with a conventional dedicated reference electrode includes a resistive return path for grounding the droplet. Without a dedicated reference electrode, the return path includes a capacitive element formed between the inactive electrode(s) and the droplet through the dielectric film (fig. 15). For this reason, in order to make this current return path effective, it is necessary to activate the electrode with a time-varying voltage. The time-varying voltage may be bipolar, in which case the high voltage signal is both positive and negative with respect to the "0V" inactive electrode. In another embodiment, the time-varying voltage may be unipolar, in which case the high voltage signal is only positive and the adjacent electrodes are driven antagonistically so that the electric field across the droplet periodically reverses direction.

Other examples of substrates for EWOD droplet driving can be found in WO2021041709, which is incorporated herein by reference in its entirety.

Some aspects of the present disclosure provide different surfaces. In some embodiments, the surface is a dielectric. In some embodiments, the surface includes a dielectric layer disposed over the one or more electrodes. In some embodiments, the surface is a surface of a polymer film. In some embodiments, the surface comprises one or more nucleotides bound to the surface. In some embodiments, the surface is a surface of a lubricating liquid layer.

Forming smooth dielectric surfaces on electrode arrays

To electrically isolate the droplets from the electrode array, a layer of dielectric may be coated on the top surface of the electrode array. The top surface of the dielectric layer may be formed to have a top surface that produces little resistance to movement of the droplet such that the droplet may move at low driving voltages (less than 100V, less than 80V, less than 50V, less than 40V, less than 30V, less than 20V, less than 15V, less than 10V, less than 8V, or less direct current, depending on smoothness, hydrophobicity, or any combination thereof). To obtain a smooth surface with low resistance, the dielectric surface may have a smooth surface morphology and may be hydrophobic or otherwise reduce the adhesion of the droplets. The chemical treatment may also be directly performed on the surface of the dielectric.

Smooth morphological surfaces are generally characterized by their roughness values. Through experimentation, it has been found that the voltage required to effect droplet motion can vary as the surface becomes smoother. The smoothness may be less than 2 μm, 1 μm, 500nm or less. An example of a method of forming a smooth dielectric surface for EWOD droplet driving can be found in WO2021041709, which is incorporated herein by reference in its entirety.

Surface chemical modification (functionalization)

Referring to fig. 1, the surface energy may be reduced by chemical modification, for example, by coating a hydrophobic or low surface energy material (840), such as a fluorocarbon-based polymer (fluoropolymer), polyethylene, polypropylene, or other hydrophobic surface coating, or the like, over the electrode (120), dielectric (130), or any combination thereof.

The surface coating may be applied using one or more methods including spin coating, dip coating, spray coating, drop coating, chemical vapor deposition, or other methods.

In some cases, it may be desirable to select a conformal coating that can be used as either a dielectric (to insulate the droplets from the charge of the electrical pad while allowing the electric field to propagate) or as a coating that is hydrophobic or hydrophilic or both (to reduce adhesion and smooth droplet movement).

Drop movement, merging and splitting

The droplets may move, coalesce, split, or any combination thereof on the open-face electrowetting device. The same principle applies to a two-plate configuration (with the droplet sandwiched in between).

In some embodiments, applying a voltage to the electrodes may make the covered surface hydrophilic, and the droplets may then wet it. When a voltage is applied across two adjacent electrodes, the droplet may spread across the two driven electrodes. When the voltage is removed from the electrode and applied to another adjacent electrode, the surface returns to the original hydrophobic state and the droplet is squeezed out. By sequentially controlling the voltages applied to the electrode grids, the position of the droplets on the surface can be precisely controlled.

In some embodiments, when two droplets are pulled toward the same electrode, the two droplets may naturally merge due to surface tension. This principle can be applied to combining multiple droplets to generate larger volumes of droplets that spread across multiple electrodes.

In some embodiments, a droplet may be divided into two smaller droplets by a sequence of voltages applied across multiple electrodes (at least three electrodes). In some embodiments, a single large droplet is consolidated on a single electrode. In some embodiments, equal voltages are applied to three adjacent electrodes simultaneously, thereby causing a single droplet to spread across the three adjacent electrodes. In some embodiments, closing the center electrode may force the droplet to move to the two outer electrodes. Since the potentials of the two outer electrodes are equal, the droplet may then be split into two smaller droplets.

Laboratory in box (Table digital laboratory wet)

Any combination of the manufacturing methods described above may be used in the applications described in this section.

In some embodiments, described herein is an apparatus that can provide a general-purpose machine that can automate a variety of biological protocols/assays/tests. The device may include a box that may have an openable and closable lid. The cover may have a transparent window to observe the movement of the droplets on an electrode array, which may be formed as a digital microfluidic chip. The cartridge may house a digital microfluidic chip capable of moving, merging, dividing droplets, where the droplets may carry biological reagents. The microfluidic chip may also have one or more heaters or coolers that are capable of heating the droplets to a temperature of up to 150 degrees celsius or higher, or cooling the droplets to a temperature of-20 degrees celsius or lower.

One aspect of the present disclosure provides a thermal control. In some embodiments, chip thermal control.

The droplets may be dispensed onto the chip by one or more "liquid dispensers". Each liquid dispenser may be, for example, an electrofluidic pump, syringe pump, simple tube, robotic pipette, spray nozzle, acoustic spray device, or other pressure or non-pressure driven device. Droplets may be fed from a reservoir labeled "kit" into a liquid dispenser. A "laboratory in a cassette" may have up to several hundred reagent cartridges that interact with the microfluidic chip directly.

Droplets can move from the digital microfluidic chip onto the microplate. The microplate may be a plate with wells that can accommodate samples. Microplates may have anywhere from one to one million wells on a single plate. A plurality of microplates may be interfaced with the chips in the cassette. For dispensing droplets from a microfluidic chip to a microplate, electrowetting chips with various geometries may be used. In some cases, the dispensing chip may be shaped like a cone of a pipette tip. In another embodiment, the dispensing opening may be a cylinder. In another embodiment, the distribution device may be two parallel plates with a gap therebetween. In another embodiment, the dispensing device may be a single open face over which at least one droplet moves. The dispensing mechanism may also use numerous other mechanisms, such as electrofluidic pumps, syringe pumps, tubing, capillaries, paper, cartridges or even simple wells of a chip, etc.

One aspect of the present disclosure provides a microfluidic dispensing chip.

The "laboratory in the box" may be climate controlled to regulate internal temperature, humidity, lighting conditions, droplet size, pressure, droplet coating, oxygen concentration, or any combination thereof. The interior of the box may be under vacuum. The interior of the box may be purged with a combination of gases. The gas may include air, argon, nitrogen or carbon dioxide.

The digital microfluidic chip located in the center of the cassette can be removed, cleaned and replaced.

The digital microfluidic chip located in the center of the cartridge may be disposable.

Digital microfluidic devices may include sensors, such as spectroscopy or acoustic sensors, for performing various assays.

The digital microfluidic device may include magnetic bead-based separation units for DNA size selection, DNA purification, protein purification, plasmid extraction, and any other biological workflow using magnetic beads. The device can perform multiple magnetic bead-based operations simultaneously on a single chip, varying from one to one million.

The box may be equipped with multiple cameras to view the chip from the top, sides and bottom. The camera may be used to locate drops on the chip, measure the volume of drops, measure mixing, and analyze ongoing reactions. Information from these sensors can be provided as feedback to a computer controlling the current to the electrodes in order to precisely control the droplets to achieve high throughput speeds by precise droplet positioning, mixing, etc. Information from these sensors may be provided to a machine learning algorithm or neural network. The machine learning data may also be used to ensure that the assigned protocol has been executed correctly. This may be accomplished by building a machine-learned classifier that automatically monitors and tracks events that occur during the trial that may be indicative of atypical events. The machine learning algorithm may also optimize the box and improve the trial. Detecting abnormal fluid phenomena in a database of operational data may help optimize test performance. This data will help to improve test results and cassette reliability.

The laboratory in the cassette may be used to perform microplate operations such as plate stamping, serial dilution, plate replication and Kong Chongpai.

The laboratory in the cassette may include equipment for PCR amplification and DNA assembly (Gibson assembly, golden Gate assembly), molecular cloning, DNA library preparation, RNA library preparation, DNA sequencing, single cell sorting, cell incubation, cell culture, cell testing, cell lysis, DNA extraction, protein extraction, RNA and cell-free protein expression.

Processing workstation

The electrowetting chip (with or without a laboratory housing in a cassette) may include one or more workstations for performing various functions.

Mixing and splitting station

In some embodiments, the electrowetting device may incorporate one or more mixing stations. In some embodiments, a 2 x 2 set of electrowetting-based hybrid workstations may operate in parallel. A single hybrid workstation may have a 3 x 3 grid of drive electrodes. Each mixing station may be used to mix biological samples, chemical reagents, and liquids. For example, droplets of two reagents may be pooled together at a mixing station and then mixed by moving the pooled droplets around eight electrodes outside a 3 x 3 grid, or by other modes for mixing the two original droplets. The center-to-center spacing between the mixing stations may be 9mm, corresponding to the spacing of a standard 96-well plate.

The mixing station can be extended to have a variety of different configurations. Each single mixer may consist of any number of drive electrodes in a x B mode. Furthermore, the spacing between mixers is arbitrary and can be varied to suit the application (such as other SDS plates). The parallel mixing station may also have any number of individual mixers in mxn mode. The parallel mixing station may have any configuration of top plate including, but not limited to, an open face, a closing plate, or a closing plate with liquid access holes.

The mixing station may be used as a singulation station. The segmentation station may use electrowetting forces to segment one droplet into a plurality of droplets. In addition to electrowetting forces, other methods of separating droplets may be used, including dielectric wetting forces, dielectrophoresis effects, acoustic forces, hydrophobic knives, or any combination thereof. The separation may be used for a variety of purposes, such as dispensing reagents or samples. The split droplets may then be mixed with other droplets to react in the other droplets. The fragmented droplets may be analyzed by the same sensor and method as the undivided droplets.

The segmented droplets may be mixed with the target droplets to maintain a constant volume of at least one target droplet that has lost volume (e.g., due to evaporation, segmentation itself, etc.). The instructions to mix the droplets may come from an accessory device such as a computer or a smart phone.

Temperature control workstation

In some embodiments, the electrowetting chip may include one or more temperature control workstations. Each workstation may integrate one or more functions applied to the liquid sample, such as mixing, heating (e.g., to 150 degrees celsius and below), cooling (e.g., to-20 degrees celsius and below), compensating for fluid loss due to evaporation, and sample temperature homogenization. Heating or cooling may be achieved by metal traces, foil heaters, peltier elements external to the substrate, or a combination thereof. In some cases, each station may be controlled to separate temperatures, e.g., -20 ℃, 25 ℃, 37 ℃ and 95 ℃, with separate heating elements, depending on the heat transfer power of the elements and the level of heat transfer between the stations.

The parallel temperature control station may be configured in any configuration identical to the parallel mixing station.

One aspect of the present disclosure provides a combined droplet temperature controlled solution.

A temperature control workstation as described herein may be configured to precisely control and manipulate the temperature applied to a liquid sample. In some embodiments, the temperature control workstation is configured to heat/cool the liquid sample from about 0.1 ℃ to about 1 ℃. In some embodiments, the temperature control workstation is configured to heat/cool the liquid sample at about 0.1 ℃ to about 0.2 ℃, about 0.1 to about 0.3 ℃, about 0.1 to about 0.4 ℃, about 0.1 to about 0.5 ℃, about 0.1 ℃ to about 0.6 ℃, about 0.1 ℃ to about 0.7 ℃, about 0.1 ℃ to about 0.8 ℃, about 0.1 ℃ to about 0.9 ℃, about 0.2 ℃ to about 0.3 ℃, about 0.2 ℃ to about 0.4 ℃, about 0.2 ℃ to about 0.5 ℃, about 0.2 ℃ to about 0.6 ℃, about 0.2 ℃ to about 0.7 ℃, about 0.2 ℃ to about 0.8 ℃, about 0.2 ℃ to about 0.9 ℃, about 0.2 ℃ to about 1 ℃, about 0.3 ℃ to about 0.4 ℃, about 0.3 ℃ to about 0.5 ℃, about 0.3 ℃ to about 0.3 ℃, about 0.1.1 ℃ to about 0.4 ℃ about 0.7.4 ℃ to about 0.0.7 ℃ to about 0.4 ℃ to about 0.0.7 ℃ to about 0.4 ℃, about 0.1 to about 0.0.4 ℃, about 0.4 ℃ to about 0.4 ℃ and about 0.1 to about 0.4 ℃ about 0.3 ℃ of the liquid sample. In some embodiments, the temperature control workstation may be configured to heat/cool the liquid sample at about 0.1 ℃, about 0.2 ℃, about 0.3 ℃, about 0.4 ℃, about 0.5 ℃, about 0.6 ℃, about 0.7 ℃, about 0.8 ℃, about 0.9 ℃, or about 1 ℃. In some embodiments, the temperature control workstation may be configured to heat/cool the liquid sample at least about 0.1 ℃, about 0.2 ℃, about 0.3 ℃, about 0.4 ℃, about 0.5 ℃, about 0.6 ℃, about 0.7 ℃, about 0.8 ℃, about 0.9 ℃, or about 1 ℃. In some embodiments, the temperature control workstation may be configured to heat/cool the liquid sample up to about 0.2 ℃, about 0.3 ℃, about 0.4 ℃, 0.5 ℃, 0.6 ℃, about 0.7 ℃, about 0.8 ℃, about 0.9 ℃, or about 1 ℃. In some embodiments, the temperature control workstation may be configured to heat/cool the liquid sample by about 0.5 ℃. In some embodiments, the temperature control workstation is configured to heat/cool the liquid sample to maintain the temperature of the liquid sample within a range of about 0.1 ℃ to about 1 ℃ of the target temperature.

Magnetic bead workstation

In some embodiments, the magnetic bead workstation may contain samples with nucleic acids, proteins, cells, buffers, magnetic beads, wash buffers, elution buffers, and other liquids on the electrode grid. The workstation may be configured to mix a sample and reagents in a sequential order, apply heat or other process to perform operations such as nucleic acid isolation, cell isolation, protein isolation, peptide purification, isolation or purification of biopolymers, immunoprecipitation, in vitro diagnostics, exosome isolation, cell activation, cell expansion, isolation, or any combination thereof, of a particular biomolecule. In addition to mixing and heating of the liquid, each magnetic bead station may have the ability to locally turn on and off a strong and varying magnetic field, thereby allowing the magnetic beads to be moved to the bottom of, for example, an electrowetting chip. Each magnetic bead station may also have the ability to remove redundant supernatant and wash fluids by electrowetting forces or by other forces.

In some cases, the sample may be placed on an open face with a single plate electrowetting device. In some cases, the sample may be sandwiched between two plates. As with the parallel mixing station described above, multiple magnetic bead stations may be configured to operate in parallel.

Aspects of the present disclosure provide solutions where the droplet or reagent includes one or more magnetic beads. In some embodiments, the first droplet or reagent comprises one or more magnetic beads. In some embodiments, the second droplet or reagent is a magnetic bead. In some embodiments, the third droplet or reagent comprises one or more magnetic beads. In some embodiments, the combined droplets or reagents comprise one or more magnetic beads.

In some embodiments, the magnetic bead station is a fixed location on the array and/or substrate that corresponds to a fixed magnet, permanent magnet, or electromagnet. In some embodiments, the arrays and/or matrices described herein are operably coupled to a movable magnet. In embodiments where the array and/or substrate is coupled to a movable magnet, the magnetic bead stations described herein may be movable along the plane of the array and/or substrate.

Movable magnet

The present disclosure provides a system for inducing droplet motion, comprising: (a) A surface configured to support the droplet, the droplet comprising at least one bead formed of a material configured to couple to a magnetic field; (b) An actuator coupled to a magnet, wherein the magnet is configured to provide the magnetic field, and wherein the actuator is configured to translate the magnetic field along a plane parallel to the surface; and (c) a controller operably coupled to the actuator, wherein the controller is configured to instruct the actuator to translate the magnetic field along the plane such that the droplet undergoes movement along the surface as the magnetic field translates along the plane. In some embodiments, the actuator is a switch. In some embodiments, the actuator includes a motor coupled to the magnet, wherein the motor is configured to translate the magnet in a direction parallel to the surface. In some embodiments, the system further comprises electrodes configured to provide an electric field to the surface, wherein the electric field and the magnetic field are sufficient to cause the movement of the droplet. In some embodiments, the actuator is configured to move the magnet to translate along at least two axes parallel to the plane. In some embodiments, the magnet comprises a permanent magnet. In some embodiments, the magnet comprises at least one electromagnet. In some embodiments, the actuator comprises a pivot, wherein the pivot is coupled to the surface. In some embodiments, the surface includes a dielectric disposed over the one or more electrodes. In some embodiments, the one or more magnets are disposed below the surface. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises a liquid having an affinity for the surface.

Fig. 37 shows an example of droplet operations using a magnetic field. Beads formed of a material configured to couple to a magnetic field are provided in droplets on a surface (fig. 37A). A magnetic field is provided to the surface and an actuator translates the magnetic field along a plane parallel to the surface and the droplet, thereby moving the microbeads outside the droplet (fig. 37B). Droplet operations may be implemented in any system of the present disclosure having a magnetic field.

Magnetic bead washing supporting EWOD

The magnetic particles can be manipulated on the surface of the chip using a controllable local magnetic field. As the magnetic particles, for example, it is possible to prepare from microspheres. The control of the local magnetic field may be achieved by, for example, placing a solenoid, a magnet, a pair of magnets, or any combination thereof, near the particles, or by generating a magnetic field within the EWOD chip. Separation and washing based on magnetic beads can be performed on an array supporting EWOD. The droplets can be manipulated using drive electrodes, which can also position the droplets. The magnetic field may be used to concentrate the magnetic particles in a small area. The liquid may be separated from the magnetic particles by EWOD-based, dielectrophoresis-based or other electrodynamic-based driving means. The separation can be performed in split and double plate systems. Since EWOD driving can be used to position the droplets, a liquid handling robot can also be used to aspirate fluid from the chip, leaving the magnetic particles on the chip surface. The removal of liquid may be achieved by one or more holes in the array, using capillary forces, aerodynamic forces, electrodynamic forces such as EWOD or dielectric wetting, or any combination thereof. The waste liquid may be collected in a reservoir located below the array. For processes involving magnetic beads, algorithms based on computer vision may be used to notify and provide feedback to the liquid processor and/or array. The treatment may include, for example, pipetting the supernatant, resuspending the beads, preventing the magnetic beads from being pipetted with the supernatant during supernatant removal, or any combination thereof.

Nucleic acid delivery workstation

In some embodiments, the electrowetting chip may include one or more nucleic acid delivery workstations. Each nucleic acid delivery workstation can be designed to insert genetic material, other nucleic acids, and biological agents into cells using a variety of insertion methods. The insertion may be performed by applying a strong electric field, applying a strong magnetic field, applying ultrasound, applying a laser beam, or other techniques. The one or more nucleic acid delivery workstations may be configured as a single station on the electrowetting device, or multiple nucleic acid delivery workstations may be provided for parallel operation.

Optical detection workstation

In some embodiments, one or more optical detection stations using optical detection and assay methods may be provided on the electrowetting device. A light source (e.g., broad spectrum light, single frequency, etc.) may be passed through the optics to condition the light (which may include, for example, filters, diffraction gratings, mirrors, etc.) and illuminate the sample on the electrowetting device. An optical detector, which may be placed on the same side or on the other side of the electrowetting device, may be configured to detect the spectrum of light passing through the sample for analysis. The optical detection may be used to measure, for example, the concentration of nucleic acid, to measure the mass of nucleic acid, to measure the density of cells, to measure the degree of mixing between two liquids, to measure the volume of a sample, to measure the fluorescence of a sample, to measure the absorbance of a sample, the quantification of a protein, a colorimetric assay, an optical assay, or any combination thereof.

In some embodiments, the sample may be placed on an open face with a single plate electrowetting device. In some embodiments, the sample may be sandwiched between two plates. In some embodiments, the electrowetting chip and the electrodes may be transparent. In some embodiments, there may be holes in the electrode where the sample is placed in order to pass light from the light source through the sample to the optical detector or in order to introduce the sample, reagents or reactants.

In some embodiments, the optical detection may be performed on samples arranged in a 2 x 2 sample format or 96 well plate format for optical detection, or on any M x N format to measure, for example, one million samples. The samples and corresponding units of measurement may be arranged in any regular and irregular format.

Liquid treatment workstation

In some embodiments, the electrowetting device may comprise one or more workstations for loading biological samples, chemical reagents and liquids from a source well, plate or reservoir onto the electrowetting chip.

In some embodiments, the droplets may be loaded onto the electrowetting surface by acoustic droplet ejection. The source plate may hold a liquid in the aperture and may be coupled to the piezoelectric sensor by an acoustic coupling fluid. Acoustic energy from the piezoelectric acoustic sensor can be focused onto the sample in the well. In some embodiments, the electrowetting chip is located on top and is inverted. Due to the additional wetting force caused by the voltage, the droplets may adhere to the electrowetting chip, which aids the droplet sorting function of the device. The droplets ejected from the orifices using acoustic energy may be attached to the upper electrowetting device or may be combined with droplets that have moved to the acoustic wave injection station.

In some embodiments, the electrowetting device may comprise one or more workstations designed to load biological samples, chemical reagents and liquids onto the electrowetting chip through a micro diaphragm pump based dispenser.

Acoustic drop ejection techniques or micro-diaphragm pumps can be used to dispense fluid drops of picoliter, nanoliter, or microliter volumes. In some embodiments, an electrowetting device placed over the source plate captures the droplets ejected from the orifice plate and holds the droplets by electrowetting forces. In this way, a sample containing, for example, biological reagents, chemical reagents, or a combination thereof, may be dispensed onto the electrowetting chip. In some embodiments, the electrowetting plate is located at the bottom and the acoustic drop ejection sensor or micro diaphragm pump is located at the top. The input valve and larger micro diaphragm pump may be used to meter the flow of fluid into the micro diaphragm pump. In this method, a dispenser may be used to place the sample on an electrowetting chip in any position.

In some cases, the electrowetting chip may be an open plate configuration (without a second plate), and the droplets may be loaded directly onto the chip. In some cases, the electrowetting chip may have a second plate sandwiching the droplet between the electrode array and the ground electrode. In some cases, the second plate (cover plate, grounded or not) may have holes to allow the droplets to pass through. In some cases, the droplets may be loaded onto the open plate first, and then a second plate may be added. In some cases, the liquid loaded onto the electrowetting chip is ready to perform a workflow when the chip is inside the acoustic liquid handler. In some cases, the liquid loaded onto the electrowetting chip is ready to perform a workflow when the chip is located outside the acoustic liquid handler or micro diaphragm pump. In some cases, the liquid is loaded onto the electrowetting chip when the workflow is performed. In some cases, an acoustic droplet injector or micro diaphragm pump may be mounted on a positionable carriage (somewhat like a 3D printer nozzle) that is movable over the electrowetting device so that droplets may be injected at specific points on the electrowetting device.

In some cases, both the source and destination may be electrowetting chips. In this case, the chip may be configured such that the electrode arrays thereof face each other. In some cases, droplets may be transferred back and forth between the top and bottom electrowetting chips using an acoustic or electric field and different wetting affinities. Here, there may be acoustic sensors and coupling fluid on both sides of the chip. In some cases, the sample on the electrowetting chip may be the source and the destination may be the well plate. Here, an acoustic drop jet may be used to transfer the sample from the electrowetting chip onto the well plate.

The spacing of the wells in the well plate from each other, and even the form of liquid loading onto (and removal from) the electrowetting chip, may be in the form of a standard well plate or any other SDS well plate or any form. The number of holes in the plate may be any number ranging from one to one million.

The electrowetting chip loaded with a sample from an acoustic droplet ejection device or a micro-membrane pump device may be combined with one or more of a mixing station, an incubation station, a magnetic bead station, a nucleic acid delivery station, an optical detection station, other functions, or any combination thereof.

Dry/freeze dried reagents on chip

The chemical reagents, biological reagents, or combinations thereof may be lyophilized/dried/spotted on the surface of the array. Reagents may be spotted on the surface of a disposable cartridge compatible with the array. The reagent may include, but is not limited to, buffers, salts, surfactants, nucleic acids, proteins, stabilizers, microbeads, enzymes, antibiotics, or any combination thereof. The reagents may be dissolved or resuspended in an appropriate solution by a liquid handling system, EWOD drive, manual pipetting, or any combination thereof. Dried reagents may be used to produce part or all of the kits for use in various molecular biological workflows/treatments. The kit may include refrigerated conditions for storage. Molecular biological processing may include, but is not limited to, preparation of nucleic acid libraries for next generation sequencing and microbiological analysis workflows (e.g., antibiotic resistant strain detection).

Vibration typeAuxiliary mixing

The droplets may be mixed in a variety of ways. The present disclosure provides methods by which vibration of a digital microfluidic surface may be utilized to facilitate mixing of liquids on a digital microfluidic device surface. Vibration can create small scale fluid motion within droplets on the surface of a digital microfluidic device. This movement may promote diffusion and rapidly accelerate the mixing process. One example of the benefit of vibration assisted droplet mixing is the efficient capture of DNA onto magnetic particles (e.g., beads) and ultimately higher yields of extracted DNA. In some embodiments, an electrowetting array comprising an open face is provided.

One common problem with digital microfluidic platforms is achieving robust mixing of various reagents and droplets. For example, highly viscous droplets can be extremely difficult to achieve efficient mixing by purely electrowetting-based motion. These types of viscous droplets are important in a variety of applications, including extraction of DNA from highly concentrated sample materials, where DNA needs to be efficiently bound to magnetic beads. In these applications, the use of purely electrowetting-based movements for mixing results in very poor mixing and thus extraction of DNA from the sample droplets.

Described herein are implementations of devices, systems, and methods for applying vibration and/or acoustic forces to a digital microfluidic surface to assist in mixing of liquids on the surface. When tuned to the proper frequency and amplitude, the vibration causes small-scale fluid movement within the droplets to promote diffusion and rapidly accelerate the mixing process. FIG. 72 shows one application of this technique for extracting DNA using magnetic beads. In fig. 72, unless vibration is utilized, the viscosity of the DNA sample may prevent efficient mixing. As a result, DNA is effectively captured on the magnetic particles, and finally, the yield of DNA extraction is improved. FIG. 72A shows suspended beads prior to vibration-assisted mixing. FIG. 72B shows the deposit of beads and DNA formed after vibration assisted mixing.

Vibration also helps to enhance the mobility of the droplets. This is especially true for droplets containing particulates. Without vibration, large particles may tend to stay at the interface between the droplet and the substrate. When these particles are present on the contact line of the droplet, they can act to fix the droplet at the specified location, thereby limiting the mobility of the droplet. The introduction of vibration helps to prevent particles from settling at the contact line, a process that greatly improves the reliability of electrowetting mobility of the droplet carrying the particles.

Vibration-based mixing has a synergistic effect with electrowetting-based mixing. While vibratory mixing is effective in dispersing particles in a portion of the droplets, it is often not effective in macro-scale mixing of the entire droplet, particularly for droplets with low contact angles with the surface. The electrowetting-based droplet mixing helps to solve this problem, and by the combined action of vibration and electrowetting, mixing of a plurality of droplets of various compositions can be achieved quickly and effectively.

The vibration frequency and amplitude need to be adjusted according to the droplet and system dynamics. The resonance dynamics of a droplet depend on a number of factors, including the volume, surface tension and viscosity of the droplet. Droplets with higher contact angles tend to exhibit greater response to vibration, while droplets that spread more easily on the surface (with lower contact angles) require greater amplitude to achieve similar mixing (fig. 73). Fig. 73 shows a case where the high contact angle droplet (fig. 73A) has a larger response to vibration than the low contact angle droplet (fig. 73B). To achieve adequate mixing of droplets using vibration, the digital microfluidic device as a whole or a portion thereof may be arbitrarily displaced in the range of several micrometers to several millimeters. A displacement between 0.1mm and 10mm is a good range for this purpose. In the vibration assisted mixing scheme described above, typical vibration frequencies are in the range of 1hz to 20 khz.

In some embodiments, other methods besides vibration may be used to mix droplets that are difficult to mix. If the re-suspension of magnetic beads requires assistance, an alternating magnetic field can be used to re-suspend and mix the magnetic beads within the droplets. This can be achieved by using a rotating permanent magnet or electromagnetic coils oriented multiaxially around the droplet. With the electrode grid itself, the droplets can be mixed by exciting resonance using an ac circuit that causes the voltage on the electrodes on which the droplets drop to oscillate. This drive may benefit from having a low impedance path to the droplet itself in order to increase the amplitude of the response.

The present disclosure provides methods of droplet mixing based on electrowetting. In some embodiments, the electrowetting-based droplet mixing includes both vibration and electrowetting. In some embodiments, vibration and electrowetting work together and mix a wide variety of droplets. In some embodiments, vibration and electrowetting of the various components can be achieved quickly and efficiently. The frequency of the electrowetting-based droplet mixing can be adjusted. The amplitude for the electrowetting-based droplet mixing can be adjusted.

Vibration of the digital microfluidic surface can be achieved in a variety of ways. In one embodiment, the surface itself may act as a spring element, whereby one end of the surface is fixed and the other end is connected to the electromechanical actuator. In some embodiments, the electromechanical actuator is an oscillating mechanism or cantilever (fig. 74). The embodiment shown in fig. 74A generates a gradient of vibration energy throughout the length of the surface. Droplets near the vibrating edge of the cantilever will experience a larger amplitude than droplets near the fixed end. For example, electromagnetic actuators, voice coil actuators, piezoelectric actuators, ultrasonic sensors, rotating eccentric masses, motor drive links, and brushed/brushless/stepper motors with oscillating link mechanisms may be used.

In another embodiment, the entire surface translates vertically (fig. 74B). This may be achieved by an electromechanical actuator comprising various flexible elements (e.g. linear flexures) or by conventional bearings. The embodiment shown in fig. 74B produces a uniform vibration amplitude across the surface (assuming a sufficiently rigid substrate is used).

The vibration mechanism described herein that limits surface motion may be driven by a number of different methods, including electromagnetic actuators, piezoelectric actuators, ultrasonic sensors, rotating eccentric masses, and brush/brushless/stepper motors with oscillating linkage mechanisms (fig. 75A).

The electromagnetic voice coil actuator can be driven over a wide frequency and amplitude range, and these can be independently controlled. In addition, various waveforms may be used to energize the actuator to achieve different effects. For example, a sine wave may be used to produce a quiet oscillation, while a rectangular wave may be used to more strongly excite a surface.

The embodiment shown in fig. 75 creates a dynamic system to be characterized and understood in order to effectively couple the driving force into the droplet vibration and ultimately achieve effective mixing (fig. 76). By using external elements such as passive springs, dampers or masses, the resulting dynamic system can be modified or enhanced. For example, these elements may be used to transform the resonant frequency of the system to a frequency that coincides with the resonant frequency of the droplets on the surface. This can be done passively or actively (with a driven spring element). In some embodiments, disposable widgets may be attached to the system between the surface and the system, the system being modified for greater stability and reliability. The widget may be a sponge clip and may promote vibration of surfaces within the system.

In some embodiments, the system may be leveled by a user through the use of a digital leveling interface. Such digital leveling can improve the function of vibration mixing and reduce the occurrence of droplet splitting caused by vibration. The leveling interface may instruct a user how to properly level the system and, by way of a leveling module configured to detect the angle of the surface, may ensure that the system has been properly leveled. In some embodiments, a voice coil actuator may be used to generate vibrations. The voice coil actuator may include a permanent magnetic field assembly and a coil assembly. Voice coil actuators can be driven over a wide range of frequencies and amplitudes. The voice coil actuator may be excited by various waveforms. For example, a sine wave may be used to produce a quiet oscillation, while a rectangular wave may be used to more strongly excite a surface.

In some embodiments, a motor-driven linkage may be used to generate vibration (fig. 75B). A conventional brushless motor, a brushed motor, or a stepping motor may be used for the rotation shaft. The shaft may be connected to a rigid or flexible link that when rotated results in an oscillating motion of the output. The motor driven links may be enhanced with, for example, passive springs, masses and damping elements to improve efficiency and allow for greater amplitude oscillations with less input power. For example, a spring element may be placed between the output of the linkage mechanism and the oscillating substrate. This embodiment is much less dependent on the surface and system dynamics of the motion constraint mechanism, but therefore may require more power input to achieve vibration output equivalent to a well tuned dynamic system.

In some embodiments, a rotating eccentric mass may be used to generate vibration. The rotating eccentric mass may be offset from the center of the rotation point. The motor may be mounted on the oscillating substrate (directly or via a coupling spring). The motor may rotate the eccentric mass to produce an oscillating acceleration. The operation of the rotating eccentric mass may result in uneven centripetal forces, which in turn may cause the motor to move back and forth. The amplitude and frequency of the acceleration may be directly related.

The resonant frequency of the system may be excited at or near the resonance peak in order to achieve optimal efficiency between the input and output power. In some embodiments, it may also be advantageous to excite the system at a location remote from the resonant frequency. This may be beneficial, for example, if it is desired to excite the droplets at substantially equal amplitudes over a wide frequency range. This special case can be easily achieved by adjusting the natural frequency of the system to a level that is reduced relative to the desired frequency range, as a result of which an almost constant acceleration amplitude is obtained over a wide frequency range (fig. 77).

In another embodiment, closed loop control may be used for fine control of frequency independent amplitude. This may be achieved, for example, by using an accelerometer mounted on the vibrating platform. The microcontroller may communicate with the sensor and calculate the acceleration amplitude in real time. Given some desired acceleration amplitude, the microcontroller can adjust the gain of the amplifier and adjust the drive waveform of the vibration actuator to precisely control the vibration amplitude.

In addition to mechanically induced vibration, in some embodiments, electrowetting or other non-contact forces may be used alone to vibrate the droplet. In some embodiments, other non-contact forces that may be used for vibration-assisted mixing include sonic or ultrasonic waves.

In some embodiments, electrowetting vibration assisted mixing may be achieved by switching the amplitude or polarity of the electric field at a specific frequency. For optimal vibration assisted mixing, the frequency should be close to the resonance frequency (or multiple of resonance frequency) of the droplet. For droplets between 10 and 200 μl, this frequency is typically in the range of 10Hz to 100 Hz. In some embodiments, only the polarity of the electric field is switched by alternately charging and discharging electrodes beneath or adjacent to the droplet while driving surrounding electrodes with signals of opposite polarity.

Some aspects of the present disclosure provide a method of producing a biopolymer, comprising: (a) Providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent; (b) Moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet, and (ii) form a combined droplet comprising the first reagent and the second reagent; and in the combined droplet, (c) forming at least a portion of the biopolymer using at least (i) the first reagent and (ii) the second reagent, wherein (b) - (c) are performed in 10 minutes or less. In some embodiments, vibration is applied to the synthetic droplets during (b), (c), or both.

One aspect of the present disclosure includes a system for processing a sample, the system comprising: an array comprising a plurality of electrodes and a surface configured to support a sample; an electromechanical actuator coupled to the array, wherein the actuator is configured to vibrate the array; and a controller operably coupled to the plurality of electrodes or the electromechanical actuator, wherein the controller is configured to instruct at least a subset of the plurality of electrodes to provide an electric field that alters the wetting characteristics of the surface or to instruct the electromechanical actuator to apply a vibration frequency to the array. In some embodiments, the controller is configured to perform (i) and (ii). In some embodiments, the controller is coupled to the plurality of electrodes and the electromechanical actuator. In some embodiments, the sample is a droplet. In some embodiments, the droplet comprises about 1 nanoliter to 1 milliliter. In some embodiments, the droplet comprises a biological material. In some embodiments, the biological sample comprises one or more biomolecules. In some embodiments, the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof. In some embodiments, the electromechanical actuator comprises a cantilever. In some implementations, the electromechanical actuator includes one or more coupling members coupled to the array. In some embodiments, the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with oscillating linkage mechanisms, or any combination thereof. In some embodiments, the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof. In some embodiments, the electromagnetic actuator comprises an electromagnetic voice coil actuator. In some embodiments, the vibration frequency comprises a gradient. In some embodiments, the gradient rises from near the point where the cantilever couples to the array. In some embodiments, the vibration has a mode. In some embodiments, the pattern is sinusoidal. In some embodiments, the pattern is square. In some embodiments, the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes. In some embodiments, the top surface includes a layer. In some embodiments, the layer comprises a liquid. In some embodiments, the layer comprises a coating. In some embodiments, the coating is hydrophobic. In some embodiments, the layer comprises a film. In some embodiments, the film is a dielectric film. In some embodiments, the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises silicone, silane, fluoropolymer treatment, parylene coating, any other suitable surface chemical modification process, ceramic, clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the first plurality of electrodes, the dielectric, the surface configured to support a droplet containing the sample, or any combination thereof may be removed from the array.

In some embodiments, the electromechanical actuator is configured to displace a surface or portion of a surface by 0.05 millimeters (mm) to 10mm. In some embodiments, the surface or the portion of the surface is displaced by about 0.05mm to about 10mm. In some embodiments of the present invention, in some embodiments, the surface or the portion of the surface is displaced from about 0.05mm to about 0.1mm, from about 0.05mm to about 0.5mm, from about 0.05mm to about 1mm, from about 0.05mm to about 2mm, from about 0.05mm to about 3mm, from about 0.05mm to about 4mm, from about 0.05mm to about 5mm, from about 0.05mm to about 6mm, from about 0.05mm to about 7mm, from about 0.05mm to about 8mm, from about 0.05mm to about 9mm, from about 0.05mm to about 10mm, from about 0.1mm to about 0.5mm, from about 0.1mm to about 1mm, from about 0.1mm to about 2mm, from about 0.1mm to about 3mm, from about 0.1mm to about 4mm about 0.1mm to about 5mm, about 0.1mm to about 6mm, about 0.1mm to about 7mm, about 0.1mm to about 8mm, about 0.1mm to about 9mm, about 0.1mm to about 10mm, about 0.5mm to about 1mm, about 0.5mm to about 2mm, about 0.5mm to about 3mm, about 0.5mm to about 4mm, about 0.5mm to about 5mm, about 0.5mm to about 6mm, about 0.5mm to about 7mm, about 0.5mm to about 8mm, about 0.5mm to about 9mm, about 0.5mm to about 10mm, about 1mm to about 2mm, about 1mm to about 3mm, about 1mm to about 4mm about 1mm to about 5mm, about 1mm to about 6mm, about 1mm to about 7mm, about 1mm to about 8mm, about 1mm to about 9mm, about 1mm to about 10mm, about 2mm to about 3mm, about 2mm to about 4mm, about 2mm to about 5mm, about 2mm to about 6mm, about 2mm to about 7mm, about 2mm to about 8mm, about 2mm to about 9mm, about 2mm to about 10mm, about 3mm to about 4mm, about 3mm to about 5mm, about 3mm to about 6mm, about 3mm to about 7mm, about 3mm to about 8mm, about 3mm to about 9mm, about 3mm to about 10mm, about 4mm to about 5mm, about 4mm to about 6mm, about 4mm to about 4mm, about 5mm to about 6mm, about 5mm to about 7mm, about 5mm to about 8mm, about 5mm to about 9mm, about 7mm to about 7mm, about 9mm to about 7mm, about 7mm to about 9mm, about 7mm to about 10mm, about 10mm to about 10mm, about 7mm to about 9 mm. In some embodiments, the surface or the portion of the surface is displaced by about 0.05mm, about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, or about 10mm. In some embodiments, the surface or the portion of the surface is displaced by at least about 0.05mm, about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, or about 8mm. In some embodiments, the surface or the portion of the surface is displaced by at most about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, or about 10mm.

In some embodiments, the frequency of the vibration is from 1 hertz (Hz) to 20 kilohertz (kHz). In some embodiments, the frequency of the vibration is about 1Hz to about 10Hz. In some embodiments, the frequency of the vibration is about 1Hz to about 2Hz, about 1Hz to about 3Hz, about 1Hz to about 4Hz, about 1Hz to about 5Hz, about 1Hz to about 6Hz, about 1Hz to about 7Hz, about 1Hz to about 8Hz, about 1Hz to about 9Hz, about 1Hz to about 10Hz, about 2Hz to about 3Hz, about 2Hz to about 4Hz, about 2Hz to about 5Hz, about 2Hz to about 6Hz, about 2Hz to about 7Hz, about 2Hz to about 8Hz, about 2Hz to about 9Hz, about 2Hz to about 10Hz, about 3Hz to about 4Hz, about 3Hz to about 5Hz, about 3Hz to about 6Hz, about 3Hz to about 8Hz, about 3Hz to about 9Hz, about 4Hz to about 5Hz, about 4Hz to about 6Hz, about 4Hz to about 7Hz, about 4Hz to about 8Hz, about 2Hz to about 9Hz, about 2Hz to about 10Hz, about 5Hz to about 10Hz, about 10Hz to about 10Hz, about 5Hz, about 7Hz to about 9Hz, about 10Hz, about 5Hz to about 7Hz, about 5Hz, about 7Hz to about 9Hz, about 10Hz, about 5Hz to about 6Hz, about 5Hz, about 7Hz, about 5Hz to about 6Hz, about 5Hz to about 9Hz. In some embodiments, the frequency of the vibration is about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the frequency of the vibration is at least about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, or about 9Hz. In some embodiments, the frequency of the vibration is at most about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the frequency of vibration is about 10Hz to about 1,000Hz. In some embodiments of the present invention, in some embodiments, the frequency of vibration is from about 10Hz to about 100Hz, from about 10Hz to about 200Hz, from about 10Hz to about 300Hz, from about 10Hz to about 400Hz, from about 10Hz to about 500Hz, from about 10Hz to about 600Hz, from about 10Hz to about 700Hz, from about 10Hz to about 800Hz, from about 10Hz to about 900Hz, from about 10Hz to about 1,000Hz, from about 100Hz to about 200Hz, from about 100Hz to about 300Hz, from about 100Hz to about 400Hz, from about 100Hz to about 500Hz, from about 100Hz to about 600Hz, from about 100Hz to about 700Hz, from about 100Hz to about 800Hz, from about 100Hz to about 900Hz, from about 100Hz to about 1,000Hz, from about 200Hz to about 300Hz, from about 200Hz to about 400Hz, from about 200Hz to about 500Hz, from about 200Hz to about 600Hz, from about 200Hz to about 700Hz, from about 200Hz to about 900Hz, from about 200Hz, from about 1,000Hz, from about 100Hz, to about 1,000Hz about 300Hz to about 400Hz, about 300Hz to about 500Hz, about 300Hz to about 600Hz, about 300Hz to about 700Hz, about 300Hz to about 800Hz, about 300Hz to about 900Hz, about 300Hz to about 1,000Hz, about 400Hz to about 500Hz, about 400Hz to about 600Hz, about 400Hz to about 700Hz, about 400Hz to about 800Hz, about 400Hz to about 900Hz, about 400Hz to about 1,000Hz, about 500Hz to about 600Hz, about 500Hz to about 700Hz, about 500Hz to about 800Hz, about 500Hz to about 900Hz, about 500Hz to about 1,000Hz, about 600Hz to about 700Hz, about 600Hz to about 800Hz, about 600Hz to about 900Hz, about 600Hz to about 1,000Hz, about 700Hz to about 800Hz, about 700Hz to about 700Hz, about 1,000Hz, about 800Hz to about 900Hz, or about 1,000Hz to about 1,000Hz. In some embodiments, the frequency of the vibration is about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the frequency of the vibration is at least about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, or about 900Hz. In some embodiments, the frequency of the vibration is up to about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the frequency of the vibration is from about 1kHz to about 20kHz. In some embodiments, the frequency of the vibration is about 1kHz to about 2.5kHz, about 1kHz to about 5kHz, about 1kHz to about 7.5kHz, about 1kHz to about 10kHz, about 1kHz to about 12.5kHz, about 1kHz to about 15kHz, about 1kHz to about 17.5kHz, about 1kHz to about 20kHz, about 2.5kHz to about 5kHz, about 2.5kHz to about 7.5kHz, about 2.5kHz to about 10kHz, about 2.5kHz to about 12.5kHz, about 2.5 to about 15kHz, about 2.5kHz to about 17.5kHz, about 2.5kHz to about 20kHz, about 5 to about 7.5kHz, about 5 to about 10kHz, about 5 to about 12.5kHz, about 5 to about 15kHz, about 5 to about 17.5kHz, about 5 to about 20kHz, about 7.5kHz to about 10kHz, about 7.5kHz to about 12.5kHz, about 7.5kHz to about 12 kHz, about 10kHz to about 10kHz, about 10.5 kHz to about 17.5kHz, about 10kHz to about 10kHz, about 10.5 to about 15kHz, about 10.5 to about 17.5kHz, about 10kHz to about 10.5 kHz, about 10.5 to about 17 kHz, about 10.5 kHz to about 15kHz, about 10.5 to about 17.5kHz, about 10.5 kHz to about 17.5kHz, about 10kHz, about 5kHz to about 17.5kHz. In some embodiments, the frequency of the vibration is about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz. In some embodiments, the frequency of the vibration is at least about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, or about 17.5kHz. In some embodiments, the frequency of the vibration is at most about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz.

Another aspect of the present disclosure includes a method for processing a sample, the method comprising: providing an array comprising a plurality of electrodes; and a surface configured to support a sample; wherein the array is coupled to an electromechanical actuator and the electromechanical actuator is configured to vibrate the array; introducing the droplets to a surface; directing the electromechanical actuator to apply a vibration frequency to the array. In some embodiments, the sample is a droplet. In some embodiments, the droplet comprises about 1 nanoliter to 1 milliliter. In some embodiments, the droplet comprises a biological material. In some embodiments, the biological sample comprises one or more biomolecules. In some embodiments, the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof. In some embodiments, the droplet comprises about 1 nanoliter to 1 milliliter. In some embodiments, the method further comprises indicating at least a subset of the plurality of electrodes to provide an electric field to alter the wetting characteristics of the surface. In some embodiments, the electromechanical actuator comprises a cantilever. In some implementations, the electromechanical actuator includes one or more coupling members coupled to the array. In some embodiments, the one or more coupling members include an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with oscillating linkage mechanisms, or any combination thereof. In some embodiments, the electromagnetic actuator comprises an electromagnetic voice coil actuator. In some embodiments, the vibration frequency comprises a gradient. In some embodiments, the gradient begins to rise from near the point where the cantilever couples to the array. In some embodiments, the vibration has a mode. In some embodiments, the pattern is sinusoidal. In some embodiments, the pattern is square. In some embodiments, the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes. In some embodiments, the surface includes a layer disposed over a dielectric, wherein the dielectric is disposed over the plurality of electrodes. In some embodiments, the layer comprises a liquid. In some embodiments, the layer comprises a coating. In some embodiments, the coating is hydrophobic. In some embodiments, the layer comprises a film. In some embodiments, the film is a dielectric film. In some embodiments, the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises silicone, silane, fluoropolymer treatment, parylene coating, any other suitable surface chemical modification process, ceramic, clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the first plurality of electrodes, the dielectric, the surface configured to support a droplet containing the sample, or any combination thereof may be removed from the array. In some embodiments, the frequency of the vibration displaces the surface or a portion of the surface from 0.05 millimeters (mm) to 10mm. In some embodiments, the frequency of the vibration is 1 hertz (Hz) to 20 kilohertz (kHz).

Another aspect of the present disclosure includes a method of contacting a first sample with a second sample, wherein the first sample is contained in a first droplet and the second sample is contained in a second droplet, the method comprising: providing an array comprising a plurality of electrodes; and a surface configured to support the first droplet and the second droplet; wherein the array is coupled to an electromechanical actuator and the electromechanical actuator is configured to vibrate the array; directing the first droplet and the second droplet to the surface; directing at least a subset of the plurality of electrodes to provide an electric field to change a wetting characteristic of the surface, thereby causing movement of the first and second droplets, wherein the movement of the first and second droplets comprises pooling the first and second droplets to produce a mixed droplet; and instructing the electromechanical actuator to apply a vibration frequency to the surface; thereby bringing the first sample into contact with the second sample. In some embodiments, the first sample, the second sample, or both comprise a viscous fluid. In some embodiments, the first sample, the second sample, or both comprise a biological sample. In some embodiments, there is a third droplet comprising a third reagent. In some embodiments, the biological sample comprises one or more biomolecules. In some embodiments, the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof. In some embodiments, the first sample, the second sample, or both comprise a reagent for a biological assay. In some embodiments, the first sample, the second sample, or both comprise one or more cell lysis reagents. In some embodiments, the one or more cell lysis reagents comprise a matrix configured to bind to a biological sample or a subset of biological samples. In some embodiments, the nucleic acid molecule comprises greater than 10 kilobases (kb), 20kb, 30kb, 40kb, or 50kb. In some embodiments, greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the biological sample is bound to the matrix. In some embodiments, the matrix is a functionalized bead. In some embodiments, the first reagent comprises one or more functionalized beads. In some embodiments, the second agent comprises one or more functionalized beads. In some embodiments, the third reagent comprises one or more functionalized beads. In some embodiments, the combination of the first, second, and/or third reagents comprises one or more functionalized beads. In some embodiments, the functionalized beads comprise one or more oligonucleotides immobilized thereto. In some embodiments, the first reagent comprises a polymerase. In some embodiments, the second agent comprises a polymerase. In some embodiments, the third reagent comprises a polymerase. In some embodiments, the combination of the first, second, and/or third reagents comprises a polymerase. In some embodiments, the first agent comprises a biomonomer. In some embodiments, the second agent comprises a biomonomer. In some embodiments, the third agent comprises a biomonomer. In some embodiments, the combination of the first, second, and/or third agents comprises a biomonomer. In some embodiments, the biomonomer is an amino acid. In some embodiments, the biomonomer is a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the substrate is a functionalized disc. In some embodiments, the first reagent comprises one or more functionalized discs. In some embodiments, the second agent comprises one or more functionalized discs. In some embodiments, the third agent comprises one or more functionalized discs. In some embodiments, the combination of the first, second, and/or third agents comprises one or more functionalized discs. In some embodiments, the functionalized disc includes one or more oligonucleotides immobilized thereto. In some embodiments, the method further comprises, after (d): altering the wetting characteristics of the surface by directing at least a subset of the plurality of electrodes to provide an electric field to induce movement of at least a portion of the mixed droplet to remove at least a portion of the mixed droplet. In some embodiments, at least a portion of the mixed droplet does not include a biological sample. In some embodiments, the method further comprises applying a magnetic field to the surface prior to or simultaneously with (e). In some embodiments, a magnetic field is used to immobilize the substrate. In some embodiments, the electromechanical actuator comprises a cantilever. In some implementations, the electromechanical actuator includes one or more coupling members coupled to the array. In some embodiments, the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with oscillating linkage mechanisms, or any combination thereof. In some embodiments, the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof. In some embodiments, the electromagnetic actuator comprises an electromagnetic voice coil actuator. In some embodiments, the vibration frequency comprises a gradient. In some embodiments, the gradient begins to rise from near the point where the cantilever couples to the array. In some embodiments, the vibration has a mode. In some embodiments, the pattern is sinusoidal. In some embodiments, the pattern is square. In some embodiments, the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes. In some embodiments, the surface includes a layer disposed over a dielectric, wherein the dielectric is disposed over a plurality of electrodes. In some embodiments, the layer comprises a liquid. In some embodiments, the layer comprises a coating. In some embodiments, the coating is hydrophobic. In some embodiments, the layer comprises a film. In some embodiments, the film is a dielectric film. In some embodiments, the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxyethyl fiber, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the first plurality of electrodes, the dielectric, the surface configured to support a droplet containing the sample, or any combination thereof may be removed from the array. In some embodiments, the frequency of the vibration displaces the surface or a portion of the surface from 0.05 millimeters (mm) to 10mm.

In some embodiments, the electromechanical actuator is configured to displace a surface or a portion of a surface by 0.05 millimeters (mm) to 10mm. In some embodiments, the surface or a portion of the surface is displaced by about 0.05mm to about 10mm. In some embodiments of the present invention, in some embodiments, the surface or portion of the surface is displaced from about 0.05mm to about 0.1mm, from about 0.05mm to about 0.5mm, from about 0.05mm to about 1mm, from about 0.05mm to about 2mm, from about 0.05mm to about 3mm, from about 0.05mm to about 4mm, from about 0.05mm to about 5mm, from about 0.05mm to about 6mm, from about 0.05mm to about 7mm, from about 0.05mm to about 8mm, from about 0.05mm to about 9mm, from about 0.05mm to about 10mm, from about 0.1mm to about 0.5mm, from about 0.1mm to about 1mm, from about 0.1mm to about 2mm, from about 0.1mm to about 3mm, from about 0.1mm to about 4mm, from about 0.1mm to about 1mm, from about 5mm, from about 0.1mm to about 6mm, from about 0.1mm to about 7mm, from about 0.1mm to about 8mm, from about 0.1mm to about 9mm, from about 1mm to about 10mm, from about 0.1mm to about 0.1mm, from about 0.5mm to about 5mm, from about 0.1mm to about 1mm, from about 1mm to about 2mm, from about 0.1mm to about 0.1mm, from about 1mm, from about 0.1mm to about 3mm, from about 1mm to about 1mm, from about 0.1mm to about 1mm, from about 1mm to about 2mm, from about 0.1mm to about 0.1mm, from about 0.1mm to about 6mm, from about 0.1mm to about 0.1mm, from about 0.1mm to about 6mm, from about 0mm to about 6mm and about 0mm about 1mm to about 5mm, about 1mm to about 6mm, about 1mm to about 7mm, about 1mm to about 8mm, about 1mm to about 9mm, about 1mm to about 10mm, about 2mm to about 3mm, about 2mm to about 4mm, about 2mm to about 5mm, about 2mm to about 6mm, about 2mm to about 7mm, about 2mm to about 8mm, about 2mm to about 9mm, about 2mm to about 10mm, about 3mm to about 4mm, about 3mm to about 5mm, about 3mm to about 6mm, about 3mm to about 7mm, about 3mm to about 8mm, about 3mm to about 9mm, about 3mm to about 10mm, about 4mm to about 5mm, about 4mm to about 6mm, about 4mm to about 4mm, about 5mm to about 6mm, about 5mm to about 7mm, about 5mm to about 8mm, about 5mm to about 9mm, about 7mm to about 7mm, about 9mm to about 7mm, about 7mm to about 9mm, about 7mm to about 10mm, about 10mm to about 10mm, about 7mm to about 9 mm. In some embodiments, the surface or the portion of the surface is displaced by about 0.05mm, about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, or about 10mm. In some embodiments, the surface or the portion of the surface is displaced by at least about 0.05mm, about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, or about 8mm. In some embodiments, the surface or the portion of the surface is displaced by at most about 0.1mm, about 0.5mm, about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, or about 10mm.

In some embodiments, the frequency of the vibration is 1 hertz (Hz) to 20 kilohertz (kHz). In some embodiments, the frequency of the vibration is 1 hertz (Hz) to 20 kilohertz (kHz). In some embodiments, the frequency of the vibration is about 1Hz to about 10Hz. In some embodiments of the present invention, in some embodiments, the frequency of vibration is about 1Hz to about 2Hz, about 1Hz to about 3Hz, about 1Hz to about 4Hz, about 1Hz to about 5Hz, about 1Hz to about 6Hz, about 1Hz to about 7Hz, about 1Hz to about 8Hz, about 1Hz to about 9Hz, about 1Hz to about 10Hz, about 2Hz to about 3Hz, about 2Hz to about 4Hz, about 2Hz to about 5Hz, about 2Hz to about 6Hz, about 2Hz to about 7Hz, about 2Hz to about 8Hz, about 2Hz to about 9Hz, about 2Hz to about 10Hz, about 3Hz to about 4Hz, about 3Hz to about 5Hz, about 3Hz to about 6Hz, about 4Hz to about 5Hz, about 4Hz to about 6Hz, about 4Hz to about 7Hz, about 4Hz to about 8Hz, about 4Hz to about 4Hz, about 9Hz, about 2Hz to about 6Hz, about 10Hz to about 10Hz, about 9Hz to about 10Hz, about 10Hz to about 5Hz, about 9Hz to about 5Hz, about 10Hz, about 7Hz to about 5Hz, about 7Hz, about 9Hz to about 5Hz, about 6Hz, about 5Hz, about 7Hz to about 6Hz, about 9Hz, about 10Hz to about 5Hz, about 9Hz, about 10Hz, about 6Hz to about 5 Hz. In some embodiments, the frequency of the vibration is about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the frequency of the vibration is at least about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, or about 9Hz. In some embodiments, the frequency of the vibration is at most about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the frequency of vibration is about 10Hz to about 1,000Hz. In some embodiments of the present invention, in some embodiments, the frequency of vibration is from about 10Hz to about 100Hz, from about 10Hz to about 200Hz, from about 10Hz to about 300Hz, from about 10Hz to about 400Hz, from about 10Hz to about 500Hz, from about 10Hz to about 600Hz, from about 10Hz to about 700Hz, from about 10Hz to about 800Hz, from about 10Hz to about 900Hz, from about 10Hz to about 1,000Hz, from about 100Hz to about 200Hz, from about 100Hz to about 300Hz, from about 100Hz to about 400Hz, from about 100Hz to about 500Hz, from about 100Hz to about 600Hz, from about 100Hz to about 700Hz, from about 100Hz to about 800Hz, from about 100Hz to about 900Hz, from about 100Hz to about 1,000Hz, from about 200Hz to about 300Hz, from about 200Hz to about 400Hz, from about 200Hz to about 500Hz, from about 200Hz to about 600Hz, from about 200Hz to about 700Hz, from about 200Hz to about 900Hz, from about 200Hz, from about 1,000Hz, from about 100Hz, to about 1,000Hz about 300Hz to about 400Hz, about 300Hz to about 500Hz, about 300Hz to about 600Hz, about 300Hz to about 700Hz, about 300Hz to about 800Hz, about 300Hz to about 900Hz, about 300Hz to about 1,000Hz, about 400Hz to about 500Hz, about 400Hz to about 600Hz, about 400Hz to about 700Hz, about 400Hz to about 800Hz, about 400Hz to about 900Hz, about 400Hz to about 1,000Hz, about 500Hz to about 600Hz, about 500Hz to about 700Hz, about 500Hz to about 800Hz, about 500Hz to about 900Hz, about 500Hz to about 1,000Hz, about 600Hz to about 700Hz, about 600Hz to about 800Hz, about 600Hz to about 900Hz, about 600Hz to about 1,000Hz, about 700Hz to about 800Hz, about 700Hz to about 700Hz, about 1,000Hz, about 800Hz to about 900Hz, or about 1,000Hz to about 1,000Hz. In some embodiments, the frequency of the vibration is about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the frequency of the vibration is at least about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, or about 900Hz. In some embodiments, the frequency of the vibration is up to about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the frequency of the vibration is from about 1kHz to about 20kHz. In some embodiments, the frequency of the vibration is about 1kHz to about 2.5kHz, about 1kHz to about 5kHz, about 1kHz to about 7.5kHz, about 1kHz to about 10kHz, about 1kHz to about 12.5kHz, about 1kHz to about 15kHz, about 1kHz to about 17.5kHz, about 1kHz to about 20kHz, about 2.5kHz to about 5kHz, about 2.5kHz to about 7.5kHz, about 2.5kHz to about 10kHz, about 2.5kHz to about 12.5kHz, about 2.5 to about 15kHz, about 2.5kHz to about 17.5kHz, about 2.5kHz to about 20kHz, about 5 to about 7.5kHz, about 5 to about 10kHz, about 5 to about 12.5kHz, about 5 to about 15kHz, about 5 to about 17.5kHz, about 5 to about 20kHz, about 7.5kHz to about 10kHz, about 7.5kHz to about 12.5kHz, about 7.5kHz to about 12 kHz, about 10kHz to about 10kHz, about 10.5 kHz to about 17.5kHz, about 10kHz to about 10kHz, about 10.5 to about 15kHz, about 10.5 to about 17.5kHz, about 10kHz to about 10.5 kHz, about 10.5 to about 17 kHz, about 10.5 kHz to about 15kHz, about 10.5 to about 17.5kHz, about 10.5 kHz to about 17.5kHz, about 10kHz, about 5kHz to about 17.5kHz. In some embodiments, the frequency of the vibration is about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz. In some embodiments, the frequency of the vibration is at least about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, or about 17.5kHz. In some embodiments, the frequency of the vibration is at most about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz.

Alternative embodiments

Drops on open face (single-plate configuration) or drops sandwiched between two plates (double-plate configuration)

For electrowetting droplet manipulation, the droplet may be placed on an open face (single plate) or sandwiched between two plates (double plate). In a two plate configuration, the droplet may be sandwiched between two plates, typically spaced 100 μm-500 μm apart. The two-plate configuration has electrodes for providing a drive voltage on one side and a reference electrode (e.g., a common ground signal) on the other side. In a two plate configuration, the continuous contact of the droplet with the reference electrode causes the electric field to create a stronger force on the droplet, thereby achieving a powerful control of the droplet. In a two-plate configuration, the droplet may be split at a lower drive voltage. In a single plate configuration, the drive electrode and the reference electrode are on the same side.

The dual plate electrowetting system can be improved using the surface treatment method described above. In a two-plate system, a droplet is sandwiched between two plates separated by a small distance. The space between the two plates may be filled with another fluid or with air only. Using the above technique to smooth the liquid-facing surfaces of the two plates to 2 μm, 1 μm or 500nm, a two-plate system can be operated at lower voltages, thereby reducing droplet residence, reducing residual trajectories, reducing cross-contamination and reducing sample loss.

Some aspects of the present disclosure provide reagents or droplets that contact a surface on only one side. In some embodiments, the first reagent or droplet is in contact with the surface on only one side. In some embodiments, the second agent or droplet contacts the surface on only one side. In some embodiments, the third reagent or droplet is in contact with the surface on only one side. In some embodiments, the combined reagents or droplets are contacted with the surface on only one side.

Electro-optical wetting (optoelectric wetting) and electro-optical wetting (photoelectric wetting)

In some embodiments, applying the potential directly to the electrode array is one way to drive the droplets using electrowetting, however, there are alternative electrowetting mechanisms other than this conventional electrowetting mechanism. Two notable mechanisms are described herein: photoelectrowetting (photoelectrowet) and photoelectrowetting (photoelectrowet), both mechanisms use light to drive the droplets. The general principles for fabricating the above-described electrowetting arrays, forming smooth surfaces and soft surfaces are applicable not only to the conventional electrowetting described above, but also to opto-electrowetting (photoelectrowet), and other forms of electrowetting.

A liquid film may be laid on the grid of photoconductors to achieve "liquid-to-liquid electrowetting". Instead of a grid of electrodes arranged under the lubricating liquid layer, either in the form of a grid of pads or as a single photoconductive circuit, the grid may be formed by a photosensitive photoconductor. The light impinging on the photoconductor may form a pattern and create an electrowetting effect. The textured solids and oils that are sufficiently transparent to light can be selected so that the underlying surface is exposed to light to create a different wetting effect.

Electrowetting (optoelectric)

In some embodiments, an photoelectrowet (optoelectric) mechanism may use a photoconductor under a conventional electrowetting circuit connected to an AC power supply. Under normal (dark) conditions, most of the impedance of the system is in the photoconductive region, so most of the voltage drop can occur there. However, when light is irradiated on the system, generation and recombination of carriers may cause the conductivity of the photoconductor to surge, and the voltage drop across the photoconductor decreases. As a result, a voltage drop occurs across the insulating layer, changing the contact angle as a function of the voltage.

Electrowetting (photoelectrowelding)

In some embodiments, electro-wetting (photo electro-wetting) is the wetting property of a surface modified with incident light (typically a hydrophobic surface). Whereas general electrowetting is observed in droplets on conductors (liquid/insulator/conductor stack) of a dielectric coating, electrowetting (photoelectric) can be observed by replacing the conductors with semiconductors (liquid/insulator/semiconductor stack).

Incident light above the bandgap of the semiconductor can generate photogenerated carriers by generating electron-hole pairs in the depletion region of the underlying semiconductor. This results in a change in the capacitance of the insulator/semiconductor laminate, resulting in a change in the contact angle of the droplet resting on the surface of the laminate. The figures illustrate the principle of the photo electro wetting effect. At zero bias (0V), the conductive droplet has a large contact angle if the insulator is hydrophobic (left figure). With increasing bias (positive for p-type semiconductor and negative for n-type semiconductor) the droplet spreads out, i.e. the contact angle decreases (center plot). In the presence of light (having energy superior to the semiconductor bandgap), the droplet spreads more due to the reduced thickness of the space charge region at the insulator/semiconductor interface.

Some aspects of the present disclosure provide a solution for exposing a reagent to light. In some embodiments, the first droplet is in light. In some embodiments, the second droplet is in light. In some embodiments, the third droplet is in light. In some embodiments, the combined droplets are in light.

Method and system for drop correction during drop operation

In one aspect, the present disclosure provides a method for processing a plurality of biological samples. The method may include receiving a plurality of droplets adjacent to an array that may contain a plurality of biological samples, and processing the plurality of biological samples in the plurality of droplets or derivatives thereof using at least the array such that a Coefficient of Variation (CV) of at least one parameter of the plurality of droplets or derivatives thereof or the array is less than 20%, such that crosstalk between the plurality of droplets is less than 5%. This may be used to process multiple biological samples. The array may be an electrowetting device as described elsewhere herein.

The at least one parameter may include one or more elements selected from droplet size, droplet volume, droplet position, droplet velocity, droplet wettability, droplet temperature, droplet pH, beads in the droplet, number of cells in the droplet, droplet color, concentration of chemical material, concentration of biological substance, or any combination thereof. The at least one parameter may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more parameters. The at least one parameter may be a measurable characteristic of the droplet.

In some embodiments, the concentration of the chemical or biological material within the droplet is monitored so as not to exceed or fall below a predetermined threshold. In some embodiments, the predetermined threshold for the concentration of the chemical or biological material is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The location may be a droplet, a reagent, a biological sample, a constituent of an array, a location of an array, a region adjacent to an array, a site of an array, or any combination thereof. The position may be corrected by at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The positions may be corrected by up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The positions may be corrected in the range of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%.

The volume of the droplet may include a volume of at least 1 picoliter (pL), 10pL, 100pL, 1 nanoliter (nL), 10nL, 100nL, 1 μl,10 μl,100 μl,1 milliliter (mL), 10mL or more. The volume of the droplet may include a volume of up to 10mL, 1mL, 100 μL,10 μL,1 μL,100 nL,10 nL, 1nL,100pL, 10pL, 1pL, or less. The volume of the droplet may be corrected by at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The volume of the droplet may be corrected up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The volume of the droplets may be corrected in the range of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%. In some embodiments, the droplet is replenished if the volume of the droplet is below a predetermined threshold. In some embodiments, the predetermined threshold may be a volume of at least 1 picoliter (pL), 10pL, 100pL, 1 nanoliter (nL), 10nL, 100nL, 1 μl,10 μl,100 μl,1 milliliter (mL), 10mL, or more. In some embodiments, the predetermined threshold may be a volume of up to 10mL, 1mL, 100 μl,10 μl,1 μl,100 nL,10 nL, 1nL,100pL, 10pL, 1pL, or less. In some embodiments, the droplet is reduced if the volume of the droplet exceeds a predetermined threshold. In some embodiments, the predetermined threshold may be a volume of at least 1 picoliter (pL), 10pL, 100pL, 1 nanoliter (nL), 10nL, 100nL, 1 μl,10 μl,100 μl,1 milliliter (mL), 10mL, or more. In some embodiments, the predetermined threshold may be a volume of up to 10mL, 1mL, 100 μl,10 μl,1 μl,100 nL,10 nL, 1nL,100pL, 10pL, 1pL, or less.

The biological sample may comprise nucleic acids, proteins, cells, salts, buffers or enzymes, wherein the droplets comprise one or more reagents for nucleic acid isolation, cell isolation, protein isolation, peptide purification, isolation or purification of biopolymers, immunoprecipitation, in vitro diagnostics, exosome isolation, cell activation, cell amplification or isolation of specific biomolecules, and wherein the droplets are subjected to reagent manipulation for nucleic acid isolation, cell isolation, protein isolation, peptide purification, isolation or purification of biopolymers, immunoprecipitation, in vitro diagnostics, exosome isolation, cell activation, cell amplification or isolation of specific biomolecules. The presence of the biological sample may be corrected by an amount of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The presence of a biological sample may be corrected by an amount of up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The presence of the biological sample may be corrected by an amount of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%.

The activity of the biological material may include enzymatic activity, cellular activity, small molecule activity, reagent activity, where the activity may be affinity, specificity, reactivity, rate, inhibition, toxicity (e.g., IC ₅₀ 、LD ₅₀ 、EC ₅₀ 、ED ₅₀ 、GI ₅₀ Etc.) or any combination thereof. The activity of a biological sample can be corrected by an amount of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99% or more. The activity of a biological sample may be corrected by an amount of up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The activity of a biological sample can be corrected by an amount of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%.

In some embodiments, the droplets have a viscosity of about 0% glycerol to about 60% glycerol at room temperature (25 ℃). In some embodiments of the present invention, in some embodiments, the droplets have about 0% to about 10% glycerol, about 0% to about 15% glycerol, about 0% to about 20% glycerol, about 0% to about 25% glycerol, about 0% to about 30% glycerol, about 0% to about 35% glycerol, about 0% to about 40% glycerol, about 0% to about 45% glycerol, about 0% to about 50% glycerol, about 0% to about 55% glycerol, about 0% to about 60% glycerol, about 10% to about 15% glycerol, about 10% to about 20% glycerol, about 10% to about 25% glycerol, about 10% to about 30% glycerol, about 10% to about 35% glycerol, about 10% to about 40% glycerol, about 10% to about 45% glycerol, about 10% to about 50% glycerol, about 10% to about 55% glycerol, and about about 10% to about 60% glycerol, about 15% to about 20% glycerol, about 15% to about 25% glycerol, about 15% to about 30% glycerol, about 15% to about 35% glycerol, about 15% to about 40% glycerol, about 15% to about 45% glycerol, about 15% to about 50% glycerol, about 15% to about 55% glycerol, about 15% to about 60% glycerol, about 20% to about 25% glycerol, about 20% to about 30% glycerol, about 20% to about 35% glycerol, about 20% to about 40% glycerol, about 20% to about 45% glycerol, about 20% to about 50% glycerol, about 20% to about 55% glycerol, about 20% to about 60% glycerol, about 25% to about 30% glycerol, about 25% to about 35% glycerol, about 25% to about 40% glycerol, about, about 25% to about 45% glycerol, about 25% to about 50% glycerol, about 25% to about 55% glycerol, about 25% to about 60% glycerol, about 30% to about 35% glycerol, about 30% to about 40% glycerol, about 30% to about 45% glycerol, about 30% to about 50% glycerol, about 30% to about 55% glycerol, about 30% to about 60% glycerol, about 35% to about 40% glycerol, about 35% to about 45% glycerol, about 35% to about 50% glycerol, about 35% to about 55% glycerol, about 35% to about 60% glycerol, about 40% to about 45% glycerol, about 40% to about 50% glycerol, about 40% to about 55% glycerol, about 40% to about 60% glycerol, about 45% to about 50% glycerol, about 45% to about 55% glycerol, about 45% to about 60% glycerol, about 50% to about 55% glycerol, or about 60% to about 60% glycerol. In some embodiments, the droplets have a viscosity of about 0% glycerol, about 10% glycerol, about 15% glycerol, about 20% glycerol, about 25% glycerol, about 30% glycerol, about 35% glycerol, about 40% glycerol, about 45% glycerol, about 50% glycerol, about 55% glycerol, or about 60% glycerol at room temperature (25 ℃). In some embodiments, the droplets have a viscosity of at least about 0% glycerol, about 10% glycerol, about 15% glycerol, about 20% glycerol, about 25% glycerol, about 30% glycerol, about 35% glycerol, about 40% glycerol, about 45% glycerol, about 50% glycerol, or about 55% glycerol at room temperature (25 ℃). In some embodiments, the droplets have a viscosity of up to about 10% glycerol, about 15% glycerol, about 20% glycerol, about 25% glycerol, about 30% glycerol, about 35% glycerol, about 40% glycerol, about 45% glycerol, about 50% glycerol, about 55% glycerol, or about 60% glycerol at room temperature (25 ℃). In some embodiments, the droplets have a viscosity of about 40% glycerol at room temperature (25 ℃).

In some embodiments, the droplets have a viscosity of about 0.1 centipoise (cP) to about 200cP at room temperature (about 25 ℃). In some embodiments of the present invention, in some embodiments, the droplets have a droplet size of about 0.1 to about 1cP, about 0.1 to about 2cP, about 0.1 to about 5cP, about 0.1 to about 10cP, about 0.1 to about 30cP, about 0.1 to about 50cP, about 0.1 to about 70cP, about 0.1 to about 100cP, about 0.1 to about 150cP, about 0.1 to about 200cP, about 1 to about 2cP, about 1 to about 5cP, about 1 to about 10cP, about about 1 to about 30cP, about 1 to about 50cP, about 1 to about 70cP, about 1 to about 100cP, about 1 to about 150cP, about 1 to about 200cP, about 2 to about 5cP, about 2 to about 10cP, about 2 to about 30cP, about 2 to about 50cP, about 2 to about 70cP, about 2 to about 100cP, about 2 to about 150cP, about 2 to about 200cP a viscosity of about 5cP to about 10cP, about 5cP to about 30cP, about 5cP to about 50cP, about 5cP to about 70cP, about 5cP to about 100cP, about 5cP to about 150cP, about 5cP to about 200cP, about 10cP to about 30cP, about 10cP to about 50cP, about 10cP to about 70cP, about 10cP to about 100cP, about 10cP to about 150cP, about 10cP to about 200cP, about 30cP to about 50cP, about 30cP to about 70cP, about 30cP to about 100cP, about 30cP to about 150cP, about 30cP to about 200cP, about 50cP to about 70cP, about 50cP to about 100cP, about 50cP to about 150cP, about 50cP to about 200cP, about 70cP to about 100cP, about 70cP to about 150cP, about 70cP to about 200cP, about 100cP to about 150cP, about 100cP to about 200cP, or about 150cP to about 200 cP. In some embodiments, the droplet has a viscosity of about 0.1cP, about 1cP, about 2cP, about 5cP, about 10cP, about 30cP, about 50cP, about 70cP, about 100cP, about 150cP, or about 200cP at room temperature (about 25 ℃). In some embodiments, the droplet has a viscosity of at least about 0.1cP, about 1cP, about 2cP, about 5cP, about 10cP, about 30cP, about 50cP, about 70cP, about 100cP, or about 150cP at room temperature (about 25 ℃). In some embodiments, the droplet has a viscosity of at most about 1cP, about 2cP, about 5cP, about 10cP, about 30cP, about 50cP, about 70cP, about 100cP, about 150cP, or about 200cP at room temperature (about 25 ℃).

In some embodiments, the droplets have a viscosity of about 0% glycerol to about 30% glycerol at room temperature (25 ℃). In some embodiments of the present invention, in some embodiments, the droplets have about 0% to about 5% glycerol, about 0% to about 7.5% glycerol, about 0% to about 10% glycerol, about 0% to about 12.5% glycerol, about 0% to about 15% glycerol, about 0% to about 17.5% glycerol, about 0% to about 20% glycerol, about 0% to about 22.5% glycerol, about 0% to about 25% glycerol, about 0% to about 27.5% glycerol, about 0% to about 30% glycerol, about 5% to about 7.5% glycerol, about 5% to about 10% glycerol, about 5% to about 12.5% glycerol, about 5% to about 15% glycerol, about 5% to about 17.5% glycerol, about 5% to about 20% glycerol, about 5% to about 22.5% glycerol, about 5% to about 25% glycerol, about 5% to about 27.5% glycerol, about 5% to about 30% glycerol, and about 15% glycerol at room temperature (25 ℃). About 7.5% to about 10% glycerol, about 7.5% to about 12.5% glycerol, about 7.5% to about 15% glycerol, about 7.5% to about 17.5% glycerol, about 7.5% to about 20% glycerol, about 7.5% to about 22.5% glycerol, about 7.5% to about 25% glycerol, about 7.5% to about 27.5% glycerol, about 7.5% to about 30% glycerol, about 10% to about 12.5% glycerol, about 10% to about 15% glycerol, about 10% to about 17.5% glycerol, about 10% to about 20% glycerol, about 10% to about 22.5% glycerol, about 10% to about 25% glycerol, about 10% to about 27.5% glycerol, about 10% to about 30% glycerol, about 12.5% to about 15% glycerol, about 12.5% to about 17.5% glycerol, about 12.5% to about 20% glycerol, about 12.5% to about 22.5% glycerol, about 12.5% to about 25% glycerol, about 12.5% to about 27.5% glycerol, about 12.5% to about 30% glycerol, about 15% to about 17.5% glycerol, about 15% to about 20% glycerol, about 15% to about 22.5% glycerol, about 15% to about 25% glycerol, about 15% to about 27.5% glycerol, about 15% to about 30% glycerol, about 17.5% to about 20% glycerol, about 17.5% to about 22.5% glycerol, about 17.5% to about 25% glycerol, about 17.5% to about 27.5% glycerol, about 17.5% to about 30% glycerol, about 20% to about 22.5% glycerol, about 20% to about 25% glycerol, about 20% to about 27.5% glycerol, about 20% to about 30% glycerol, about 22.5% to about 25% to about 25.5% glycerol, about 22.5% to about 25% to about 27.5% glycerol, about 27% to about 25% glycerol, or about 27.5% to about 25% glycerol. In some embodiments, the droplets have a viscosity of about 0% glycerol, about 5% glycerol, about 7.5% glycerol, about 10% glycerol, about 12.5% glycerol, about 15% glycerol, about 17.5% glycerol, about 20% glycerol, about 22.5% glycerol, about 25% glycerol, about 27.5% glycerol, or about 30% glycerol at room temperature (25 ℃). In some embodiments, the droplets have a viscosity of at least about 0% glycerol, about 5% glycerol, about 7.5% glycerol, about 10% glycerol, about 12.5% glycerol, about 15% glycerol, about 17.5% glycerol, about 20% glycerol, about 22.5% glycerol, about 25% glycerol, or about 27.5% glycerol at room temperature (25 ℃). In some embodiments, the droplets have a viscosity of up to about 5% glycerol, about 7.5% glycerol, about 10% glycerol, about 12.5% glycerol, about 15% glycerol, about 17.5% glycerol, about 20% glycerol, about 22.5% glycerol, about 25% glycerol, about 27.5% glycerol, or about 30% glycerol at room temperature (25 ℃).

In some embodiments, the droplets have a viscosity of about 0.5cP to about 15cP at room temperature (25 ℃). In some embodiments of the present invention, in some embodiments, the droplets have about 0.5 to about 1, about 0.5 to about 2, about 0.5 to about 3, about 0.5 to about 4, about 0.5 to about 5, about 0.5 to about 7, about 0.5 to about 9, about 0.5 to about 11, about 0.5 to about 13, about 0.5 to about 15, about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 7, about 1 to about 9, about 1 to about 11, about 1 to about 13, about 2 to about 7, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 7, about 2 to about 9, about 2 to about 11, about 2 to about 13, about 2 to about 15, about 1 to about 15 viscosity of about 3cP to about 4cP, about 3cP to about 5cP, about 3cP to about 7cP, about 3cP to about 9cP, about 3cP to about 11cP, about 3cP to about 13cP, about 3cP to about 15cP, about 4cP to about 5cP, about 4cP to about 7cP, about 4cP to about 9cP, about 4cP to about 11cP, about 4cP to about 13cP, about 4cP to about 15cP, about 5cP to about 7cP, about 5cP to about 9cP, about 5cP to about 11cP, about 5cP to about 13cP, about 5cP to about 15cP, about 7cP to about 9cP, about 7cP to about 11cP, about 7cP to about 13cP, about 7cP to about 15cP, about 9cP to about 11cP, about 9cP to about 13cP, about 9cP to about 15cP, about 11cP to about 13cP, about 11cP to about 15cP, or about 13cP to about 15 cP. In some embodiments, the droplet has a viscosity of about 0.5cP, about 1cP, about 2cP, about 3cP, about 4cP, about 5cP, about 7cP, about 9cP, about 11cP, about 13cP, or about 15cP at room temperature (25 ℃). In some embodiments, the droplet has a viscosity of at least about 0.5cP, about 1cP, about 2cP, about 3cP, about 4cP, about 5cP, about 7cP, about 9cP, about 11cP, or about 13cP at room temperature (25 ℃). In some embodiments, the droplet has a viscosity of at most about 1cP, about 2cP, about 3cP, about 4cP, about 5cP, about 7cP, about 9cP, about 11cP, about 13cP, or about 15cP at room temperature (25 ℃).

The droplet radius may be at least 0.0001 μm, 0.001 μm, 0.01 μm, 0.1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 500 μm, 1000 μm, 5000 μm, 10,000 μm, 50,000 μm, 100,000 μm or more. The droplet radius may be a maximum of 100,000 μm, 50,000 μm, 10,000 μm, 5000 μm, 1000 μm, 500 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm, 0.001 μm or less. The droplet radius may be 1000 μm to 0.0001 μm, 500 μm to 0.01 μm or 100 μm to 1 μm. The droplet radius may be corrected by an amount of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The droplet radius may be corrected by an amount up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The droplet radius may be corrected in an amount of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%.

In some embodiments, the droplet is replenished if the size of the droplet is below a predetermined threshold. In some embodiments, the droplet is reduced if the size of the droplet exceeds a predetermined threshold. In some embodiments, the predetermined threshold may be a radius of at least 0.0001 μm, 0.001 μm, 0.01 μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 500 μm, 1000 μm, 5000 μm, 10,000 μm, 50,000 μm, 100,000 μm or more. In some embodiments, the predetermined threshold may be a volume of at most 100,000 μm, 50,000 μm, 10,000 μm, 5000 μm, 1000 μm, 500 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm, 0.001 μm, or 0.0001 μm or less.

The droplet shape may be flat, annular, spherical, elliptical, oval, circular, or any combination thereof. The drop shape can be corrected to any shape. The droplets may be corrected to be flat, annular, spherical, elliptical, oval, circular, or any combination thereof.

The droplet height may be at least 0.0001 μm, 0.001 μm, 0.01 μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 500 μm, 1000 μm, 5000 μm, 10,000 μm, 50,000 μm, 100,000 μm or more. The drop height may be a maximum of 100,000 μm, 50,000 μm, 10,000 μm, 5,000 μm, 1000 μm, 500 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm, 0.001 μm, 0.0001 μm or less. The droplet height may be 1000 μm to 0.0001 μm, 500 μm to 0.01 μm or 100 μm to 1 μm. The drop height may be corrected by an amount of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The drop height may be corrected by an amount of up to 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. The drop height may be corrected in an amount of 0.001% to 20%, 0.01% to 10%, 0.01% to 5%, or 0.1% to 1%.

In some embodiments, the pH of the droplet is monitored by one or more methods disclosed herein. In some embodiments, the pH of the droplet is maintained within a predetermined threshold. In some embodiments, the pH of the droplet is maintained so as not to exceed 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the pH of the droplet is maintained at a value not less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

In some embodiments, the relative humidity level achieved is about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, the relative humidity level achieved is from about 89% to about 100%. In some embodiments of the present invention, in some embodiments, the relative humidity levels achieved are about 89% to about 90%, about 89% to about 91%, about 89% to about 92%, about 89% to about 93%, about 89% to about 94%, about 89% to about 95%, about 89% to about 96%, about 89% to about 97%, about 89% to about 98%, about 89% to about 99%, about 89% to about 100%, about 90% to about 91%, about 90% to about 92%, about 90% to about 93%, about 90% to about 94%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 90% to about 100%, about 91% to about 92%, about 91% to about 93%, about 91% to about 94%, about 91% to about 95%, about 91% to about 96%, about 91% to about 97%, about 91% to about 98%, about 91% to about 99%, about 91% to about 100%, about 100% to about 100%. About 92% to about 93%, about 92% to about 94%, about 92% to about 95%, about 92% to about 96%, about 92% to about 97%, about 92% to about 98%, about 92% to about 99%, about 92% to about 100%, about 93% to about 94%, about 93% to about 95%, about 93% to about 96%, about 93% to about 97%, about 93% to about 98%, about 93% to about 99%, about 93% to about 100%, about 94% to about 95%, about 94% to about 96%, about 94% to about 97%, about 94% to about 98%, about 94% to about 99%, about 94% to about 100%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 100%, about 97% to about 98%, about 97% to about 99%, about 99% to about 96% About 97% to about 100%, about 98% to about 99%, about 98% to about 100%, or about 99% to about 100%. In some embodiments, the relative humidity level achieved is about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the relative humidity level achieved is at least about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the relative humidity level achieved is at most about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

In addition to controlling evaporation, the heating array can also be used to precisely control drop temperature. The droplets may be heated on the open face using heaters embedded above or below the array substrate. Without some form of environmental control, large temperature differentials may occur between the internal droplet temperatures of these matrix heaters and the temperature of the heater surface. These large temperature differences can lead to inaccurate drop temperature control and can be subject to large temperature fluctuations due to factors such as ambient air flow. Furthermore, without ambient temperature control, the difference between the heater temperature and the drop temperature may constitute a function of parameters including, for example, drop surface area to volume ratio, drop size, and temperature set point. These enclosures may be completely sealed to prevent the escape of heated humid air, but may also be partially open. For example, such a design may control condensation in a cool temperature environment.

An example of a method of forming a smooth dielectric surface for EWOD droplet driving can be found in WO2021041709, which is incorporated herein by reference in its entirety.

Array without dedicated reference electrode

In some aspects of the disclosure, the arrays described herein do not include one or more dedicated reference electrodes. In these aspects, EWOD may be induced by using one or more adjacent electrode/electrodes adjacent to the drive electrode(s) as a current return path.

Aspects of the disclosure include a system for processing droplets, the system comprising: an array comprising a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded; and a surface configured to support a droplet containing a sample; a controller operably coupled to the plurality of electrodes, wherein the controller is configured to activate at least a subset of the plurality of electrodes with a time-varying voltage to change the wetting characteristics of the surface. In some embodiments, the system does not include a cover electrode. In some embodiments, the plurality of electrodes includes at least one electrode having a cross-section or overlap with the droplet sufficient to form a current return path in the vicinity of the electrode and adjacent electrodes. In some embodiments, the plurality of electrodes are coplanar. In some embodiments, the time-varying voltage is bipolar. In some embodiments, the time-varying voltage is from about 1Hz to about 20kHz.

In some embodiments, an oil layer such as silicone oil may be used as the hydrophobic coating and as the reference electrode when grounded (fig. 3A). The oil layer may be slightly conductive or polar. The dielectric surface may comprise microstructures introduced by methods including, but not limited to, the methods described herein. These microstructures can draw oil by capillary action and can be connected to ground potential by, for example, a temporarily grounded drive electrode, a dedicated ground electrode, a dedicated connection elsewhere on the array, or any combination thereof.

In some embodiments, ionized air may surround droplets within the array (fig. 3B). Ionized air may be used in the array as a reference electrode for electrowetting driving. Ionized air may be introduced by an ionized air blower and directed toward the droplets. Due to the charging (e.g., pinning) of the surface or droplet, the droplet may permanently adhere to a location. Pinning of the droplet may be slowed by neutralizing the droplet with ions introduced by the blower.

In some embodiments, the time-varying voltage is 1 hertz (hz) to 20 kilohertz (khz). In some embodiments, the time-varying voltage is 1 hertz (hz) to 20 kilohertz (khz). In some embodiments, the time-varying voltage is from about 1Hz to about 10Hz. In some embodiments, the time-varying voltage is about 1Hz to about 2Hz, about 1Hz to about 3Hz, about 1Hz to about 4Hz, about 1Hz to about 5Hz, about 1Hz to about 6Hz, about 1Hz to about 7Hz, about 1Hz to about 8Hz, about 1Hz to about 9Hz, about 1Hz to about 10Hz, about 2Hz to about 3Hz, about 2Hz to about 4Hz, about 2Hz to about 5Hz, about 2Hz to about 6Hz, about 2Hz to about 7Hz, about 2Hz to about 8Hz, about 2Hz to about 9Hz, about 2Hz to about 10Hz, about 3Hz to about 4Hz, about 3Hz to about 5Hz, about 3Hz to about 6Hz, about 3Hz to about 7Hz, about 4Hz to about 6Hz, about 4Hz to about 7Hz, about 4Hz to about 8Hz, about 4Hz to about 9Hz, about 2Hz to about 10Hz, about 3Hz to about 7Hz, about 5Hz to about 10Hz, about 5Hz to about 7Hz, about 5Hz, about 7Hz to about 10Hz, about 7Hz, about 5Hz to about 7Hz, about 6Hz, about 5Hz to about 9Hz, about 10Hz, about 5Hz, about 7Hz to about 10Hz. In some embodiments, the time-varying voltage is about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the time-varying voltage is at least about 1Hz, about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, or about 9Hz. In some embodiments, the time-varying voltage is at most about 2Hz, about 3Hz, about 4Hz, about 5Hz, about 6Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz. In some embodiments, the time-varying voltage is from about 10Hz to about 1,000Hz. In some embodiments of the present invention, in some embodiments, the time-varying voltage is about 10 to about 100Hz, about 10 to about 200Hz, about 10 to about 300Hz, about 10 to about 400Hz, about 10 to about 500Hz, about 10 to about 600Hz, about 10 to about 700Hz, about 10 to about 800Hz, about 10 to about 900Hz, about 10 to about 1,000Hz, about 100 to about 200Hz, about 100 to about 300Hz, about 100 to about 400Hz, about 100 to about 500Hz, about 100 to about 600Hz, about 100 to about 700Hz, about 100 to about 800Hz, about 100 to about 900Hz, about 100 to about 1,000Hz, about 200 to about 300Hz, about 200 to about 400Hz, about 200 to about 500Hz, about 200 to about 600Hz, about 200 to about 700Hz, about 200 to about 800Hz, about 200 to about 900Hz, about 200 to about 1,000Hz, about 300 to about 400Hz, about 400Hz about 300Hz to about 500Hz, about 300Hz to about 600Hz, about 300Hz to about 700Hz, about 300Hz to about 800Hz, about 300Hz to about 900Hz, about 300Hz to about 1,000Hz, about 400Hz to about 500Hz, about 400Hz to about 600Hz, about 400Hz to about 700Hz, about 400Hz to about 800Hz, about 400Hz to about 900Hz, about 400Hz to about 1,000Hz, about 500Hz to about 600Hz, about 500Hz to about 700Hz, about 500Hz to about 800Hz, about 500Hz to about 900Hz, about 500Hz to about 1,000Hz, about 600Hz to about 700Hz, about 600Hz to about 800Hz, about 600Hz to about 900Hz, about 600Hz to about 1,000Hz, about 700Hz to about 800Hz, about 700Hz to about 900Hz, about 700Hz to about 1,000Hz, about 800Hz to about 900Hz, or about 800Hz to about 1,000Hz. In some embodiments, the time-varying voltage is about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the time-varying voltage is at least about 10Hz, about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, or about 900Hz. In some embodiments, the time-varying voltage is up to about 100Hz, about 200Hz, about 300Hz, about 400Hz, about 500Hz, about 600Hz, about 700Hz, about 800Hz, about 900Hz, or about 1,000Hz. In some embodiments, the time-varying voltage is from about 1kHz to about 20kHz. In some embodiments of the present invention, in some embodiments, the time-varying voltage is about 1kHz to about 2.5kHz, about 1kHz to about 5kHz, about 1kHz to about 7.5kHz, about 1kHz to about 10kHz, about 1kHz to about 12.5kHz, about 1kHz to about 15kHz, about 1kHz to about 17.5kHz, about 1kHz to about 20kHz, about 2.5kHz to about 5kHz, about 2.5kHz to about 7.5kHz, about 2.5kHz to about 10kHz, about 2.5kHz to about 12.5kHz, about 2.5kHz to about 15kHz, about 2.5kHz to about 17.5kHz, about 2.5kHz to about 20kHz, about 5kHz to about 7.5kHz, about 5kHz to about 10kHz, about 5 to about 12.5kHz, about 5kHz to about 15kHz, about 5kHz to about 20kHz, about 7.5kHz to about 10kHz, about 7.5kHz to about 12 kHz, about 7.5kHz to about 12.5kHz, about 7.5kHz to about 10kHz, about 10kHz to about 10kHz, about 15kHz to about 10.5 kHz, about 10.5 kHz to about 10kHz, about 10.5 kHz to about 10.5 kHz, about 10.5 kHz or about 10.5 kHz to about 10.5 kHz. In some embodiments, the time-varying voltage is about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz. In some embodiments, the time-varying voltage is at least about 1kHz, about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, or about 17.5kHz. In some embodiments, the time-varying voltage is at most about 2.5kHz, about 5kHz, about 7.5kHz, about 10kHz, about 12.5kHz, about 15kHz, about 17.5kHz, or about 20kHz.

In some embodiments, upon activating at least a subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and the one or more inactive electrodes. In some embodiments, activation of at least a subset of the plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes. In some embodiments, the system further comprises a dielectric layer. In some embodiments, the dielectric layer includes a thickness, wherein the thickness is sufficient to ground a current generated by the plurality of electrodes. In some embodiments, the thickness is from 0.025 micrometers (μm) to 10,000 μm.

In some embodiments, the dielectric layer has a thickness of about 0.025 μm to about 10,000 μm. In some embodiments of the present invention, in some embodiments, the thickness of the dielectric layer is about 0.025 μm to about 0.05 μm, about 0.025 μm to about 0.1 μm, about 0.025 μm to about 1 μm, about 0.025 μm to about 10 μm, about 0.025 μm to about 50 μm, about 0.025 μm to about 100 μm, about 0.025 μm to about 200 μm, about 0.025 μm to about 500 μm, about 0.025 μm to about 1,000 μm, about 0.025 μm to about 5,000 μm, about 0.025 μm to about 10,000 μm, about 0.05 μm to about 0.1 μm, about 0.05 μm to about 1 μm, about 0.05 μm to about 10 μm, about 0.05 μm to about 50 μm, about 0.05 μm to about 100 μm, about 0.05 μm to about 200 μm, about 0.05 μm to about 500 μm, about 0.025 μm to about 1,000 μm, about 0.05 μm to about 1.1 μm, about 0.05 μm to about 1 μm, about 0.05 μm to about 0.1 μm, about 0.05 μm to about 100 μm, about 0.05 μm to about 200 μm about 0.1 μm to about 100 μm, about 0.1 μm to about 200 μm, about 0.1 μm to about 500 μm, about 0.1 μm to about 1,000 μm, about 0.1 μm to about 5,000 μm, about 0.1 μm to about 10,000 μm, about 1 μm to about 10 μm, about 1 μm to about 50 μm, about 1 μm to about 100 μm, about 1 μm to about 200 μm, about 1 μm to about 500 μm, about 1 μm to about 1,000 μm, about 1 μm to about 5,000 μm about 1 μm to about 10,000 μm, about 10 μm to about 50 μm, about 10 μm to about 100 μm, about 10 μm to about 200 μm, about 10 μm to about 500 μm, about 10 μm to about 1,000 μm, about 10 μm to about 5,000 μm, about 10 μm to about 10,000 μm, about 50 μm to about 100 μm, about 50 μm to about 200 μm, about 50 μm to about 500 μm, about 50 μm to about 1,000 μm, about 50 μm to about 5,000 μm, about 50 μm to about 10,000 μm, about 100 μm to about 200 μm, about 100 μm to about 500 μm, about 100 μm to about 1,000 μm, about 100 μm to about 5,000 μm, about 100 μm to about 10,000 μm, about 200 μm to about 500 μm, about 200 μm to about 1,000 μm, about 200 μm to about 5,000 μm, about 200 μm to about 10,000 μm, about 500 μm to about 1,000 μm, about 500 μm to about 5,000 μm, about 500 μm to about 10,000 μm, about 1,000 μm to about 5,000 μm, about 1,000 μm to about 10,000 μm, or about 5,000 μm to about 10,000 μm. In some embodiments, the dielectric layer has a thickness of about 0.025 μm, about 0.05 μm, about 0.1 μm, about 1 μm, about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 500 μm, about 1,000 μm, about 5,000 μm, or about 10,000 μm. In some embodiments, the dielectric layer has a thickness of at least about 0.025 μm, about 0.05 μm, about 0.1 μm, about 1 μm, about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 500 μm, about 1,000 μm, or about 5,000 μm. In some embodiments, the dielectric layer has a thickness of at most about 0.05 μm, about 0.1 μm, about 1 μm, about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 500 μm, about 1,000 μm, about 5,000 μm, or about 10,000 μm.

In some embodiments, the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the system further comprises a liquid disposed in the gap adjacent the dielectric layer and the plurality of electrodes. In some embodiments, the liquid forms an adhesion between the plurality of electrodes and the dielectric layer. In some embodiments, the liquid comprises a dielectric material. In some embodiments, the liquid prevents or reduces the conductivity of air disposed in the gap. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

Other embodiments of this aspect of the disclosure include a system for processing droplets, the system comprising: an array comprising a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded; and a surface configured to support a droplet containing a sample; a controller operably coupled to the plurality of electrodes, wherein the controller is configured to activate at least a subset of the plurality of electrodes with a voltage to change a wetting characteristic of the surface; wherein the array does not include a permanent reference electrode. In some embodiments, the voltage is a time-varying voltage. In some embodiments, the system does not include a cover electrode. In some embodiments, the plurality of electrodes includes at least one electrode having a cross-section or overlap with the droplet sufficient to form a current return path in the vicinity of the electrode and adjacent electrodes. In some embodiments, the plurality of electrodes are coplanar. In some embodiments, the time-varying voltage is bipolar. In some embodiments, the time-varying voltage is from about 1Hz to about 20kHz.

In some embodiments, upon activating at least a subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and the one or more inactive electrodes. In some embodiments, activation of at least a subset of the plurality of electrodes forms an antagonistic current drive scheme in one or more adjacent electrodes. In some embodiments, the system further comprises a dielectric layer. In some embodiments, the dielectric layer includes a thickness, wherein the thickness is sufficient to ground a current generated by the plurality of electrodes. In some embodiments, the thickness is from 0.025 micrometers (μm) to 10,000 μm.

Another embodiment of this aspect of the disclosure includes a method for moving a droplet over an array, wherein the array includes a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded, and a surface configured to support a droplet containing a sample; the method comprises the following steps: activating at least a subset of the plurality of electrodes with a time-varying voltage to alter the wetting characteristics of the surface; wherein the time-varying voltage creates a current return path adjacent the droplet and one or more inactive electrodes, thereby causing movement of the droplet. In some embodiments, the plurality of electrodes are coplanar. In some embodiments, the time-varying voltage is bipolar. In some embodiments, the time-varying voltage is from about 1Hz to about 20kHz.

In some embodiments, upon activating at least a subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and the one or more inactive electrodes. In some embodiments, activation of at least a subset of the plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

Another embodiment of this aspect of the disclosure includes a method for moving a droplet on an array, wherein the array includes a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded, and a surface configured to support a droplet containing a sample; the method comprises the following steps: activating at least a subset of the plurality of electrodes with a voltage to change a wetting characteristic of the surface; wherein the array does not include a permanent reference electrode. Wherein the time-varying voltage creates a current return path in the vicinity of the droplet and the one or more inactive electrodes, thereby causing movement of the droplet. In some embodiments, the plurality of electrodes are coplanar. In some embodiments, the time-varying voltage is bipolar. In some embodiments, the time-varying voltage is from about 1Hz to about 20kHz.

Disposable cartridge

Various methods by which the EWOD platform can be used with replaceable cartridges and/or upper surface films are described herein. Alternative, flexible or combinations thereof structures, such as thin films or membranes, enable the reuse of the drive and/or reference electrodes. The replaceable cartridge may also eliminate cross-contamination between samples in other experiments or in the same experiment. The disposable cartridge-type structure may include a dielectric, a hydrophobic layer, a reference electrode, an inlet for introducing and removing fluid, an outlet, or any combination thereof. The replaceable structure may be permanently bonded to the array. The structure may be bonded to the drive electrode using an adhesive, heat, application of vacuum, a strong electrostatic field, or any combination thereof. An example of a disposable cartridge for EWOD droplet actuation can be found in WO2021041709, which is incorporated herein by reference in its entirety.

Bulk sample handling

In some embodiments, processing a large volume sample (e.g., in microliter, centiliter, or milliliter scale) can be performed by dividing or grading a starting material (e.g., a biological sample) into aliquots with a dispenser, and then introducing the aliquots into a processing zone of the array. The input material may be processed as droplets in parallel or sequentially on the array. The input material may be, for example, a biological sample (e.g., blood, tissue, or plasma) or an environmental sample (e.g., water or soil). Sample processing on the array may include, for example, extraction of nucleic acids (e.g., DNA, RNA), isolation of specific cell types (e.g., immune cell subtypes, circulating tumor cells, or cells isolated from tissue biopsies), or isolation of extracellular vesicles (e.g., exosomes).

Array scaling

Multiplexed execution

In some embodiments, the number of drive signals may be reduced to extend from a single array tile to a large number of array tiles (e.g., 10, 20, 30, 40, 50, 100, 500 or more array tiles), and samples processed in parallel (e.g., 96 samples processed simultaneously on 96 separate tiles). For example, a common drive signal may be used to simultaneously energize electrodes on multiple chips. In addition, the reference electrodes on each sheet may be driven by separate signals. At any given time, activating a reference electrode on a particular sheet may move a droplet on that sheet, while droplets on other (e.g., inactive) sheets may not be affected by the electrodynamic force.

An arrangement comprising a plurality of reconfigurable array tiles stacked adjacent to one another in a reconfigurable tray may provide for customization of the number of tiles that are activated at run-time. The assembly may allow a single sheet or a column of sheets to be loaded in a reconfigurable tray. The reconfigurable tray, tray and sheet may be of any shape. Multiple trays may be loaded onto the reconfigurable tray to process, for example, 8, 96, 384, 1,536, 6,144, 24,576 or more samples in parallel. The trays, trays and sheets may be stacked vertically, horizontally or a combination of both.

One aspect of the present disclosure provides a microfluidic dispensing chip. Another aspect of the present disclosure provides a dispensing attachment. In some embodiments, the dispensing accessory is a chip tray. In some embodiments, the dispensing fitment is a wash station. In some embodiments, the dispensing accessory is a bar code dispensing head.

Individual control and overall control of evaporation

The evaporation of one or more droplets (samples) on a conditioning array may be achieved by processing multiple samples on an array or multiple arrays. By encapsulating individual droplets onto an array sheet using the methods described herein, large scale processing can be achieved. The entire array sheet or multiple array sheets may be covered to enclose one or more droplets simultaneously. The cover member may be lowered onto the array before, during and/or after droplet processing.

General reagent dispenser

In processing a sample, the same set of reagents (e.g., biological samples, chemical reagents, solutions, nucleic acids (e.g., DNA, RNA, PNA, etc.), optical reagents, etc.) may be introduced onto one or more patches of the array. The introduction of such reagents may use a shared dispenser that dispenses the reagents on-chip. These dispensers may include the dispensing mechanisms described herein. The dispenser may include one or more different channels. Each of the different channels may be used to dispense a single reagent throughout a given process. The dispenser may comprise only a single channel. A single channel may be used to dispense various reagents in a single process. The cleaning solution may be used to clean a single channel between dispensing different reagents to prevent any possible cross-contamination. The dispensers described herein may also be used to aspirate samples/reagents from the array surface. The washing step may be performed between successive pumping steps.

An array or arrays may be configured within a liquid handling automation instrument as described herein. The sample and reagent may be dispensed onto the array by a liquid handler. The array or arrays may be removed (e.g., manually or automatically) from the liquid handler and placed adjacent to the liquid handler.

Single sample to multiple samples

The methods and systems described herein can be used to perform a two-step process of developing and deploying biological and chemical automation workflows on an array. The workflow may be developed on a single array element and the reaction may be iterated (e.g., manually or automatically). The optimized workflow may be deployed across multiple arrays. For example, a Next Generation Sequencing (NGS) sample preparation workflow of a single sample processing unit may be developed. Subsequently, the developed single NGS sample preparation workflow may be deployed on an array capable of processing 96 samples in parallel, each of the 96 samples being processed according to the developed single NGS sample preparation workflow.

Film composite material

To prevent charge accumulation in the droplet, a patterned electrode may be used on the droplet-facing surface of the dielectric substrate. The patterned electrode can be manufactured using a variety of different manufacturing methods, including screen printing, flexographic printing, gravure printing, ink jet printing, sputtering, and vapor deposition techniques. The metallic ink used in the printing process plays an important role in determining the performance of the printed electrode. Silver particle inks can generally produce feature sizes as small as about 100 μm and have a typical minimum deposition thickness of about 1 μm.

If a thin (typically <1 μm) conformal hydrophobic coating is used to make the hydrophobic layer of the coated stack, the thickness of the printed electrode is important to determine if the droplet can move freely on the surface or be immobilized in a specified position. It is generally desirable that the trace height of the printed feature be substantially less than the droplet itself. For droplets of 100 μl or less, a 1 μm thick track with a thin hydrophobic coating can greatly impede movement.

Thus, when a thin conformal hydrophobic coating is used, it is desirable to pattern electrodes that are substantially thinner than 1 μm, as shown in fig. 13A and 13B. Particle-free ink formulations that utilize chemical reactions to precipitate metal particles can achieve smaller feature sizes (-5 μm) and produce thinner trajectories (< 100 nm). These inks can be patterned using conventional printing processes and are compatible with a variety of substrates including PET and PI dielectrics. Fig. 13A shows a droplet 10210 to be transported through an array. In some embodiments, the array includes a first layer of electrodes 10220 adjacent to a substrate 10205. A dielectric layer 10240 may be disposed over the first layer electrode 10220. A second layer electrode 10225 may be disposed over the dielectric layer 10240. A conformal coating 10235 can be disposed on top of the second layer electrode. In some embodiments, the conformal coating is hydrophobic. If the electrodes are too thick (e.g., made by some screen printing method), pinning features 10230 that resist droplet movement may be created. Thus, as shown in fig. 13B, the electrodes can be printed by the methods disclosed herein to create a particle-free electrode layer 10227 that does not impede droplet motion.

In some embodiments, the configurations described herein may be applied to the cartridges described in the several sections above.

Film frame to sheet application

In one embodiment of the electrowetting device, a thin (< 5 μm) porous membrane may be used to form a liquid injection surface where one droplet may move freely. The porous film may be adhered to the dielectric film by using a film frame in which three layers (dielectric, porous, frame) are attached to the periphery of the frame. Such adhesion may be achieved with wet adhesives, dry adhesives or by thermal lamination. These adhesion strategies may be selectively applied over regions (e.g., along the perimeter of the frame) or over the entire surface of the film.

When a combination of certain materials is used, thermal lamination may be performed. The dielectric film may be composed of PET, FEP, or PFA for thermal lamination with a textured porous film (e.g., PTFE porous film). This thermal lamination process produces a strong film that is capable of maintaining a porous top surface upon which liquid can be infused to create a liquid infused surface, thereby achieving high performance droplet mobility.

In order to achieve consistent droplet mobility across the electrode array, the film or coating stack must maintain consistent intimate contact with the electrode array and the substrate. Such intimate contact may be achieved using a variety of methods. The stretching device may be used to stretch the film-like coating to ensure intimate contact with the substrate. Alternatively, vacuum pressure may be used to tightly adhere the film to the substrate through small holes or porous features in the substrate.

As shown in fig. 14A and 14B, a matrix 10305 including electrodes 10320 may be provided. In some embodiments, a membrane 10335 may be provided over the electrode 10320 and held in place by a membrane frame 10330. When the membrane is applied, bubbles 10355 may be generated between the membrane 10335 and the electrode array 10320. These bubbles can be easily pushed to the edge of the film using a scraper or brush. In some embodiments, as shown in fig. 14B, a filler fluid 10350 is used to ensure good adhesion between the film layer and the substrate. A thin layer of fill fluid 10350 may be placed between electrode array 10320 and bottom film layer 10335 to smooth out any wrinkles in the film and remove any air gaps by surface tension. The filler liquid may be various insulating materials including silicone oil or fluorinated oil.

Use of fill fluid adjacent to electrodes in an array

In addition to embodiments that include removable arrays and/or cartridges, the fill fluid may provide adhesion between the electrodes and the rest of the array disclosed herein, and the fill fluid adjacent to the electrodes of the array described herein may provide additional advantages. For example, when filling the air gap between any two adjacent electrodes, the filling liquid acts as a high dielectric breakdown material and prevents air breakdown. Air typically has a breakdown voltage of about 1 kilovolt per millimeter. Thus, while reducing the gap between two adjacent electrodes advantageously allows for a smooth transition of the droplet, if the gap between two electrodes is reduced, conduction will begin at some point, resulting in an improper operation of the electrowetting device. By adding a fill fluid to fill the gap between the two electrodes, the gap between the electrodes can be reduced while still maintaining the operability of the array at high voltages to achieve reliable droplet motion. In some embodiments, the filler fluid is a liquid. Furthermore, in some embodiments, particularly in embodiments where the array includes a liquid layer disposed on a surface of the dielectric, the fill liquid may be a different composition than the liquid layer disposed on the surface of the dielectric. One aspect of the present disclosure includes a system for processing a sample, the system comprising: a plurality of electrodes; a dielectric layer disposed over the plurality of electrodes, wherein the dielectric layer comprises a surface configured to support a droplet comprising the sample; a liquid disposed in the gap adjacent to the plurality of electrodes and the dielectric layer. In some embodiments, the liquid creates adhesion between the plurality of electrodes and the dielectric layer. In some embodiments, the liquid comprises a dielectric material. In some embodiments, the liquid prevents or reduces the conductivity of air disposed in the gap. In some embodiments, the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises silicone, silane, fluoropolymer treatment, parylene coating, any other suitable surface chemical modification process, ceramic, clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the dielectric layer is removable. In some embodiments, the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof. In some embodiments, the synthetic polymeric material includes polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methylcellulose, hydroxypropyl methylcellulose (HPMC), ethylhydroxyethyl cellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof. In some embodiments, the fluorinated material includes Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof. In some embodiments, the liquid comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, greases, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof. In some embodiments, the dielectric layer is removable. In some embodiments, the adhesion is sufficient to secure the liquid to the surface, and wherein the liquid is resistant to gravity. In some embodiments, the liquid is selected to preferentially wet the surface to facilitate movement of the liquid droplet on the surface.

Monitoring droplets

The present disclosure provides methods for monitoring at least one droplet on an electrowetting array. The droplets may operate on an electrowetting array. The droplets may be in a reactive state.

An example of a method for monitoring droplets on a surface for EWOD droplet driving may be found in WO2021041709, which is incorporated herein by reference in its entirety.

Method for nucleic acid analysis

Isolation and transfer of High Molecular Weight (HMW) nucleic acids

The length of the complete genomic DNA may be greater than about 100 megabases (Mb), but the isolation protocol may fragment the genomic DNA into fragments ranging from 10 to 200 kilobases (kb) in length. However, low yields of intact genomic DNA molecules (e.g., >100 kb) are an unresolved limitation of DNA isolation techniques, as sequencing techniques are capable of handling longer read lengths (e.g., greater than about 1 Mb).

The HMW nucleic acid may be extracted from, for example, whole blood, serum, or saliva. HMW nucleic acid can be extracted from intact cells. The nucleic acid may be DNA. The nucleic acid may be RNA. The extracted nucleic acids may be used in a variety of applications including, for example, library preparation, amplification, sequencing, polymerase Chain Reaction (PCR), gel electrophoresis, and other processing.

Described herein are systems and methods that minimize mechanical disruption of nucleic acids (e.g., DNA) (e.g., due to shear forces of air displacement pipetting). Systems and methods are described herein that reduce sample loss due to, for example, dead volumes of conventional processing devices. The systems and methods described herein are capable of automating the isolation of high throughput and High Molecular Weight (HMW) DNA, wherein the median size of the DNA fragments is at least about 1kb, 10kb, 100kb, 1,000kb, 10,000kb, 100,000kb, 1,000,000kb, or greater. The systems and methods described herein are capable of automating the isolation of high throughput and high molecular weight DNA, wherein the median size of the DNA fragments is up to about 1,000,000kb, 100,000kb, 10,000kb, 1,000kb, 100kb, 10kb, 1kb or less. The systems and methods described herein are capable of automating the isolation of high throughput and high molecular weight DNA, wherein the DNA fragments have a median size of from about 1kb to about 1,000,000kb, from 100kb to about 500,000kb, or from about 1,000kb to about 100,000kb.

Systems and methods for whole blood extraction on an electrowetting array are described herein. In some embodiments, at least about 10 μl to at least about 500 μl of whole blood is used for extraction. In some embodiments, at least about 100 μl of whole blood is used for extraction. In some embodiments, about 10 μl to about 250 μl. In some embodiments, at least 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or about 150. In some embodiments, up to about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 500, about 450, about 500, or more μl of whole blood is used for extraction. In some embodiments, at least about 0.2 up to about 5 μg of DNA is extracted from 100 μl of blood. In some embodiments, at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 μg of DNA is extracted from 100 μl of blood.

Described herein are universal open dielectric electrowetting on (EWOD) systems and methods that can manipulate reaction volumes suitable for HMW DNA isolation. By integrating capabilities such as magnetic bead separation and heater/cooler in the same system, the systems and methods described herein may not include custom instrumentation. The systems and methods described herein may provide for direct reprogramming to expand the number of executable workflows to achieve new formulations, e.g., programmable control of variable inputs, reagents, incubation, washing steps, and thousands of drops on a single device.

The systems described herein can manipulate droplets on at least two configurations of 2D or 3D electrode grids (e.g., droplets sandwiched between two plates separated by a small gap or on an open face). For example, in a two-plate PDM system (e.g., electrode size 25 μm), one 5pL droplet may be aliquoted, transported, and mixed with another droplet. On the open face, droplets (e.g., about 200 μl) of EWOD devices (e.g., electrodes of 2mm in size) can be manipulated. The systems described herein can handle volumes suitable for, e.g., large DNA extraction (e.g., 100 μl to 1 mL) as well as droplets small enough to encapsulate single cells and nuclei (e.g., 50 nL).

In addition, in order to increase the yield of HMW DNA from the cell sample, enhanced agitation techniques may be employed on the array. The agitation technique may include methods such as mechanical buzzers, shakers, vortexers, sonication, or any combination thereof. A magnetic micro-stirrer may be introduced into the sample to enhance mixing. These agitators may be combined with the different magnet configurations described herein. Magnets of different shapes may be used to alter the shape and diffusion form of the magnetic beads on the array. Magnets with adjustable strength can be used to accommodate magnetic beads operating on an array.

DNA extracted from cells in a stabilizing buffer can produce intact HMW DNA. For example, alginate hydrogels can be used as scaffold materials for stabilizing HMW DNA. Alginate can form stable gels in the presence of cations, gelling conditions can be mild, and the gelling process can be reversed by, for example, extracting calcium ions (e.g., by adding citrate or EDTA). The extracted DNA may be stabilized in a high viscosity/low shear solution (e.g., alginate droplets) formed on the chip. This stabilization method allows the HMW genomic DNA to be transferred without significant degradation (e.g., in a laboratory or between different sites). The HMW DNA can be stored in an agent (e.g., alginate hydrogel) to prevent shearing. The extracted HMW DNA can be transferred to a tube, for example, after extraction, or stored on an EWOD array. To prevent DNA cleavage prior to sequencing, the sequencing library can be assembled on the same device used for HMW DNA extraction. Likewise, nanopores may also be integrated into the array for direct sequencing without sample transfer.

Sample preparation

Annular sample preparation

The present disclosure provides methods of sample preparation for sequencing. The method can include performing High Molecular Weight (HMW) nucleic acid extraction. The method may further comprise sample preparation. The sample preparation method may produce a circular nucleic acid. In one example, the nucleic acid may be deoxyribonucleic acid (DNA). The method of sample preparation may be cyclization or recycling. In one example, the addition of a polymerase and/or ligase can convert the double stranded nucleic acid into a circular form. Nucleic acids can be circularized by covalent closure of the DNA "sticky" ends. Nucleic acids may be circularized by recombination between redundant terminal sequences. The nucleic acid may be circularized by binding proteins at the ends of the viral DNA. Nucleic acids can be circularized on an electrowetting array.

The present disclosure provides methods of preparing a sample on an electrowetting array. The sample preparation method may comprise preparing a sample for sequencing. The sample preparation method may comprise preparing a sample for Circular Consensus Sequencing (CCS). The sample preparation method may be cyclization. The sample preparation method may comprise preparing a sample for Rolling Circle Amplification (RCA). The nucleic acid may be circularized by providing a droplet adjacent to the electrowetting array, wherein the droplet comprises the nucleic acid. Nucleic acids can be circularized by combining a droplet with one or more reagent droplets. The one or more reagents may be reagents for circularizing a nucleic acid sample. The one or more reagents may be enzymes. Examples of enzymes may include polymerases and ligases. In one example, a single polymerase may be used to sequence a nucleic acid sample.

The nucleic acid may be circularized by treating the droplets using an electrowetting array to circularize the nucleic acid. The circularized nucleic acid sample may be separated from one or more reagent droplets. The sample droplet may be combined with one or more reagent droplets and subsequently separated from the one or more reagent droplets. Subsequently, one or more reagent droplets may be combined with the second droplet. The method may further include performing one or more droplet operations on the electrowetting array to treat the droplet, wherein the one or more droplet operations include contacting one or more reagent droplets with the droplet. Examples of nucleic acid circularization are also provided in "Template Preparation and Sequencing Guide" of Pacific Biosciences of Califorinia (see https:// www.paccom/wp-content/uploads/2015/09/Guide-Pacific-Bioscien cesTemplate-Preparation-and-sequencing. Pdf) and WO2009120374, the entire contents of which are incorporated herein by reference. Nucleic acid sample preparation methods can produce high sequencing reads. Nucleic acid sample preparation methods can produce sequencing reads that are at least about 70 kilobases (kb) in length. The nucleic acid sample preparation method can produce a sequencing read of at least about 80kb in length. The nucleic acid sample preparation method can produce a sequencing read of at least about 200kb in length. The method of nucleic acid sample preparation can result in a sequencing read of at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, or at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb, or more in length. In one example, the nucleic acid sample preparation method may be performed on an electrowetting array. In another example, the nucleic acid may be deoxyribonucleic acid (DNA).

The nucleic acid sample preparation method can produce sequencing data of at least about 100 gigabytes (Gb). The nucleic acid sample preparation method can produce data of at least about 10 Gb. The nucleic acid sample preparation method can produce sequencing data of at least about 30 Gb. The nucleic acid sample preparation method can produce sequencing data of at least about 500 Gb. The nucleic acid sample preparation method can produce sequencing data of at least about 512 Gb. The nucleic acid sample preparation method can produce at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or greater sequencing data.

The circularized nucleic acid sample may comprise a plurality of sequences comprising said target sequence. At least about 80% of the plurality of sequences may comprise the target sequence. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of the plurality of sequences may comprise the target sequence.

The nucleic acid sample preparation methods disclosed herein can provide a high purity nucleic acid sample. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). The nucleic acid sample preparation methods disclosed herein can provide at least one sequencing read having an a260/a280 ratio of up to about 1.93. The nucleic acid sample preparation methods disclosed herein can provide at least one sequencing read with an a260/a280 ratio of up to about 1.84. The nucleic acid sample preparation methods disclosed herein can provide at least one sequencing read having an a260/a280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, or less.

The present disclosure provides a method of producing a circularized nucleic acid sample having a longer insert size comprising (a) providing a droplet adjacent to an electrowetting array, the droplet comprising a nucleic acid sample, (b) treating the droplet with the electrowetting array to circularize the nucleic acid sample, and (c) subjecting the circularized nucleic acid sample to a sequencing reaction using a single polymerase. The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for circularizing the nucleic acid sample. The method may further comprise combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. The method may further include performing one or more droplet operations on the electrowetting array to treat the droplet, wherein the one or more droplet operations include contacting one or more reagent droplets with the droplet. Methods that produce circularized nucleic acid samples with longer insert sizes can produce high sequencing reads. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing reads having a length of at least about 70 kilobases (kb). Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing reads having a length of at least about 80 kb. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing reads having a length of at least about 200 kb.

Methods of producing circularized nucleic acid samples having a longer insert size can produce sequencing reads of at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb or longer in length.

In one example, the method of generating circularized nucleic acid samples having longer insert sizes can be performed on an electrowetting array. The nucleic acid may be deoxyribonucleic acid (DNA). Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing data of at least about 100 gigabytes (Gb). Methods of producing circularized nucleic acid samples having longer insert sizes can produce data of at least about 10 Gb. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing data of at least about 30 Gb. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing data of at least about 500 Gb. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing data of at least about 512 Gb. Methods of producing circularized nucleic acid samples having longer insert sizes can produce sequencing data of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or greater.

The circularized nucleic acid sample may comprise a plurality of sequences comprising a target sequence. At least about 80% of the plurality of sequences may comprise the target sequence. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of the plurality of sequences may comprise the target sequence.

Methods of producing circularized nucleic acid samples having longer insert sizes can provide nucleic acid samples having high purity. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). Methods of producing circularized nucleic acid samples having longer insert sizes can provide at least one sequencing read having an A260/A280 ratio of up to about 1.93. Methods of producing circularized nucleic acid samples having longer insert sizes can provide at least one sequencing read having an A260/A280 ratio of up to about 1.84. Methods of producing circularized nucleic acid samples having longer insert sizes can provide at least one sequencing read having an A260/A280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1 or less.

The present disclosure provides a method for circularizing a nucleic acid sample, comprising: providing a droplet adjacent to an electrowetting array, wherein the droplet comprises the nucleic acid sample; combining the droplets with one or more reagent droplets; treating the droplets using an electrowetting array to circularize the nucleic acid sample; separating the droplets from the one or more reagent droplets; and combining the one or more reagent droplets with the sample droplet to generate a circularized nucleic acid sample. The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for circularizing the nucleic acid sample. The method may further comprise combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. The method may further comprise performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting one or more reagent droplets with the droplets.

The method of circularizing nucleic acid samples can produce sequencing reads that are at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb, or longer in length.

In one example, the method of circularizing a nucleic acid sample can be performed on an electrowetting array. The nucleic acid may be deoxyribonucleic acid (DNA).

Methods of circularizing nucleic acid samples can produce sequencing data of at least about 100 gigabytes (Gb). The method of circularizing a nucleic acid sample can produce data of at least about 10 Gb. Methods of circularizing nucleic acid samples can produce sequencing data of at least about 30 Gb. Methods of circularizing nucleic acid samples can produce sequencing data of at least about 500 Gb. Methods of circularizing nucleic acid samples can produce sequencing data of at least about 512 Gb. The method of circularizing a nucleic acid sample can produce at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or greater sequencing data.

Methods of circularizing nucleic acid samples can provide high purity nucleic acid samples. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). The method of circularizing a nucleic acid sample can provide at least one sequencing read having an a260/a280 ratio of up to about 1.9. The method of circularizing a nucleic acid sample can provide at least one sequencing read having an a260/a280 ratio of up to about 1.84. The method of circularizing a nucleic acid sample can provide at least one sequencing read having an a260/a280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.93, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1 or less.

The present disclosure provides a method for generating a sequencing library comprising (a) providing a nucleic acid sample comprising a plurality of nucleic acid molecules, the nucleic acid molecules comprising a plurality of sequences; (b) Generating a sequencing library using the nucleic acid sample, wherein the sequencing library comprises at least 80% of a plurality of sequences of its complement; (c) Processing the droplets using the electrowetting array to circularize the nucleic acid sample; (d) Separating the droplets from the one or more reagent droplets; and (e) combining the one or more reagent droplets with the sample droplet to generate a circularized nucleic acid sample. The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for circularizing the nucleic acid sample. The method may further comprise combining the sample droplet with the one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. The method may further include performing one or more droplet operations on the electrowetting array to treat a droplet, wherein the one or more droplet operations include contacting the one or more reagent droplets with the droplet.

Methods of generating sequencing libraries can produce sequencing reads that are at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb, or longer in length. In one example, the method of generating sequencing library sizes can be performed on an electrowetting array. The nucleic acid may be deoxyribonucleic acid (DNA).

Methods of generating sequencing libraries can produce sequencing data of at least about 100 gigabytes (Gb). Methods of generating sequencing libraries can produce data of at least about 10 Gb. Methods of generating sequencing libraries can produce sequencing data of at least about 30 Gb. Methods of generating sequencing libraries can produce sequencing data of at least about 500 Gb. Methods of generating sequencing libraries can produce sequencing data of at least about 512 Gb. Methods of generating a sequencing library can produce sequencing data of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or more.

Methods of generating sequencing library sizes can provide nucleic acid samples with high purity. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). The method of generating a sequencing library may provide at least one sequencing read having an a260/a280 ratio of up to about 1.93. The method of generating a sequencing library may provide at least one sequencing read having an a260/a280 ratio of up to about 1.84. Methods of generating sequencing libraries can provide at least one sequencing read having an a260/a280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, or less.

Next Generation Sequencing (NGS) sample preparation

The systems and methods described herein may enable complete digitization for high-throughput automation of NGS sample preparation. A whole genome sequencing (WSG) library can be prepared starting from purified DNA using the systems and methods described herein. For example, DNA can be enzymatically fragmented, end repaired, and a-overhangs added on the arrays described herein. Double-indexed barcodes can be attached to DNA fragments and the size of the final ligation product can be purified and selected by magnetic bead-based purification. The method may be performed on a single device as described herein. In one example, library preparation includes ligating aptamers at both ends of a nucleic acid. Examples of sample preparation for NGS include, but are not limited to, cluster generation techniques by Illumina corporation (see "Technology Spotlight: illumina Sequencing Technology," which is incorporated herein by reference in its entirety). Sample preparation methods for NGS may be advantageous for Whole Genome Sequencing (WGS). Sample preparation methods for NGS may include the use of hybridization capture. Sample preparation methods for NGS may include the use of Unique Molecular Identifiers (UMIs) to improve sensitivity and sequencing.

The present disclosure provides methods of preparing a sample on an electrowetting array. The sample preparation method may comprise preparing a sample for sequencing. The sample preparation method may include preparing a sample for NGS. The method may include providing a sample droplet and at least one reagent droplet. The method may further comprise using a droplet operation on the electrowetting array to bring the sample droplet into contact with the at least one reagent droplet. The one or more reagent droplets may include one or more reagents for performing NGS sample preparation. The one or more reagents may be part of a NovaSeq 6000 Kit (see "NovaSeq 6000Reagent Kit", the entire contents of which are incorporated herein by reference), for example, illumina. Bridge amplification may be performed in preparation for sequencing. Sample preparation of NGS may be performed on an electrowetting array with a single tube scheme. Sample preparation of NGS may be performed on an electrowetting array by an automated method. Sample preparation for NGS may be performed on an electrowetting array for at least about 1 to at least about 14 days. Sample preparation for NGS may be performed on an electrowetting array for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. Sample preparation for NGS may produce at least about 1 to at least about 10 μg of DNA. Sample preparation for NGS may produce at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more μg of DNA. Sample preparation for NGS may result in a read length of at least about 10 to at least about 500 kb. Sample preparation for NGS may result in a read length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500kb or more. Sample preparation for NGS may include the use of inserts of various sizes. Sample preparation for NGS may include the use of inserts ranging in size from about 100bp to about 600 bp. Sample preparation for NGS may include using an insert of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600bp or longer in size. Sample preparation for NGS may include the use of inserts ranging in size from about 300 to about 450 bp. Sample preparation for NGS can produce libraries of various sizes. Sample preparation for NGS can produce libraries of about 100bp to about 600bp in size. Sample preparation for NGS may result in library sizes of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600bp or longer. Sample preparation for NGS may include the use of libraries about 400 to about 600bp in size.

The present disclosure provides systems and methods for sample preparation for nanopore sequencing. In some embodiments, nanopore sequencing may include performing one-step real-time PCR (RT-PCR) on the nucleic acid, followed by sequencing the nucleic acid on a nanopore device. Sample preparation may be performed on an electrowetting array by operating with droplets on the electrowetting array to bring at least one sample droplet into contact with at least one reagent droplet. Sample preparation for nanopore sequencing on an electrowetting array can yield high N50, or sequence length of the shortest contig that is 50% of the total genome length. Sample preparation for nanopore sequencing on an electrowetting array can yield an N50 of at least about 10kb to at least about 50 kb. Sample preparation for nanopore sequencing on an electrowetting array can yield N50 of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50kb or longer. Sample preparation for nanopore sequencing on an electrowetting array can yield nucleic acids with higher Kong Zhanyou rates. Sample preparation for nanopore sequencing on an electrowetting array can produce nucleic acids with high pore occupancy of at least about 24 hours. Sample preparation for nanopore sequencing on an electrowetting array can yield nucleic acids of high pore occupancy of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more hours. Examples of Nanopore sequencing include, but are not limited to, nanopore's MinION sequencing (see MinION's product description; lu, hengyun, francesca Giordano and Zemin Ning. "Oxford Nanopore MinION sequencing and genome assembly." Genomics, proteomics & bioinformation 14.5 (2016): 265-279; the entire contents of which are incorporated herein by reference).

Amplification of

The present invention provides a sample preparation method for amplification. Amplification may provide for sequencing. Amplification may involve binding of the initiator protein to double stranded nucleic acid. The initiation protein may bind to the 5' end of one strand of the double-stranded nucleic acid. The starter protein may introduce at least one gap in the double stranded nucleic acid. Helicase-dependent amplification may occur in which the helicase helices the double stranded nucleic acid and at least one single stranded binding protein coats at least one strand of the double stranded nucleic acid. The process may be single chain binding (SSB) protein dependent amplification. The nucleic acid may be deoxyribonucleic acid (DNA). The amplification method may comprise Rolling Circle Amplification (RCA). The amplification method may comprise bridge amplification.

RCA

The present disclosure provides a method of performing amplification. Amplification may provide for sequencing. Amplification may involve binding of the initiator protein to double stranded nucleic acid. The initiation protein may bind to the 5' end of one strand of the double-stranded nucleic acid. The starter protein may introduce at least one gap in the double stranded nucleic acid. Helicase-dependent amplification may occur in which the helicase helices the double stranded nucleic acid and at least one single stranded binding protein coats at least one strand of the double stranded nucleic acid. The process may be single chain binding (SSB) protein dependent amplification. The nucleic acid may be deoxyribonucleic acid (DNA). The amplification method may comprise Rolling Circle Amplification (RCA).

Examples of rolling circle amplification include, but are not limited to, the protocol for circularizing nucleic acid described in U.S. patent No. 9,290,800; U.S. patent No. 11,067,562; U.S. publication No. US20190360997; pacBIO SMRT sequencing (see https:// www.pacb.com/smart-science/smart-sequence ncing /); wenger, aaron M. Et al, "Accurate circular consensus long-read sequencing improves variant detection and assembly of a human geno me." Nature biotechnology 37.10.10 (2019): 1155-1162; cirSeq of Illumina (see https:// www.illumina.com/science/sequencing-method-explorer/kits-and-arrays/CirSeq. Html); acevedo, a., andio r., "Library preparation for highly accurate population sequencing of RNA viruses.," Nat protoc.2014jul;9 (7) 1760-9); hunt, m., silva, n.d., otto, t.d., et al, "circulator: automated circularization of Genome assemblies using long sequenci ng ready," Genome Biol 16,294 (2015); and Wilson, brandon D et al, "High-Fidelity Nanopore Sequencing of Ultra-Short DNATarges." Ana lytical chemistry, volume 91, 10 (2019): 6783-6789, the entire contents of which are incorporated herein by reference.

The amplification method may be performed on an electrowetting array. The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for circularizing the nucleic acid sample. The one or more reagent droplets may include one or more reagents that perform Rolling Circle Amplification (RCA). The one or more reagents may be enzymes. Examples of enzymes may include nicking enzymes, DNA polymerases, and RCR proteins. The one or more reagents may be control nucleic acids, buffer solutions and/or saline solutions, including, for example, divalent metal ions, i.e., mg ²⁺ 、Mn ²⁺ 、Ca ²⁺ And/or Fe ²⁺ . The one or more reagents may produce single stranded nucleic acids.

Methods of performing amplification can result in sequencing reads that are at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb, or longer in length. In one example, the method of performing amplification may be performed on an electrowetting array. The nucleic acid may be deoxyribonucleic acid (DNA).

Methods of performing amplification can produce at least about 100 gigabytes (Gb) of sequencing data. Methods of performing amplification can produce data of at least about 10 Gb. Methods of performing amplification can produce sequencing data of at least about 30 Gb. Methods of performing RCA can produce sequencing data of at least about 500 Gb. Methods of performing amplification can produce sequencing data of at least about 512 Gb. Methods of performing amplification can produce sequencing data of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or greater.

The circularized nucleic acid sample may comprise a plurality of sequences comprising said target sequence. At least about 80% of the plurality of sequences may comprise the target sequence. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of the plurality of sequences may comprise the target amplification.

Methods of performing amplification can provide high purity nucleic acid samples. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). The method of performing amplification may provide at least one sequencing read having an a260/a280 ratio of up to about 1.93. The method of performing amplification may provide at least one sequencing read having an a260/a280 ratio of up to about 1.84. Methods of performing amplification can provide at least one sequencing read having an a260/a280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1 or less.

Bridge amplification

The present disclosure provides a method of performing amplification. Amplification may provide for sequencing. Amplification may include bridge amplification. Double-stranded nucleic acid molecules may be provided and denatured. The original template of the denatured double-stranded nucleic acid molecule may be washed away, thereby producing a single-stranded nucleic acid molecule. The single stranded nucleic acid molecule may be covalently attached to the flow cell surface. The single-stranded molecule may form a bridge. A second strand complementary to the bridge may be formed, thereby creating a double stranded nucleic acid bridge. The second strand may be formed using a polymerase. The two strands can then be separated to produce two separate single stranded nucleic acid molecules. This process may be repeated. Some chains may be removed from the flow cell surface. The 3' -end of the remaining strand may be blocked. The sequencing primer may hybridize to the aptamer sequence of each remaining strand. Examples of bridge amplification include, but are not limited to, cluster generation techniques from Illumina corporation (see "Technology Spotlight: illumina Sequencing Technology," which is incorporated herein by reference in its entirety).

The present disclosure provides a method of bridge amplification on an electrowetting array. The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for performing bridge amplification. The one or more reagents may be part of a NovaSeq 6000 Kit (see "NovaSeq 6000Reagent Kit", the entire contents of which are incorporated herein by reference), for example, illumina. Bridge amplification may be performed in preparation for sequencing.

Sequencing

Circular consensus sequencing

The present invention provides methods of sequencing a nucleic acid sample. The method of sequencing can generate long reads. The method of sequencing can generate long high fidelity (HiFi) reads. The method of sequencing may include multiple cycle sequencing of a single template molecule. In one example, the method of sequencing may be circular consensus sequencing. The sequencing reaction may include multiple cycle sequencing. Each cycle of sequencing can generate at least one sequencing read. One or more sub-reads of the sequencing reads may be generated. Consensus sequences can be generated from sub-reads of the sequencing reads.

Examples of circular consensus sequencing are described by Wenger, aaron M. Et al, "Accurate circul ar consensus long-read sequencing improves variant detection and asse mbly of a human genome" -Nature biotechnology 37.10.37.10 (2019): 1155-1162; cirSeq of Illumina (see https:// www.illumina.com/science/sequencer ng-method-explorer/kits-and-arrays/CirSeq. Html); acevedo, a., andio r., "Library preparation for highly accurate population sequencing of RN aviruses.," Nat protoc.2014jul;9 (7) 1760-9); hunt, m., silva, n.d., otto, t.d., et al, "circulator: automated circularization of Genome assemblie s using long sequencing ready," Genome Biol 16,294 (2015); and Wi lson, brandon D et al, "High-Fidelity Nanopore Sequencing of Ultra-Shor t DNA targets." Analytical chemistry, volume 91, 10 (2019): 6783-6789; U.S. patent No. 7,906,284; U.S. patent No. 10,563,255; and WO2009120374, the entire contents of which are incorporated herein by reference

The present disclosure provides a method of sequencing a nucleic acid sample comprising (a) providing a droplet adjacent to an electrowetting array, the droplet comprising a nucleic acid sample, (b) treating the droplet with the electrowetting array to circularize the nucleic acid sample, and (c) subjecting the circularized nucleic acid sample to a sequencing reaction using a single polymerase.

The method of sequencing may further comprise combining the sample droplet with one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. The method may further include performing one or more droplet operations on the electrowetting array to treat the droplet, wherein the one or more droplet operations include contacting one or more reagent droplets with the droplet.

The electrowetting array may also include one or more reagent droplets. The one or more reagent droplets may include one or more reagents for circularizing the nucleic acid sample. The one or more reagent droplets may include one or more reagents that perform Rolling Circle Amplification (RCA). The one or more reagents may be enzymes. Examples of enzymes may include nicking enzymes, DNA polymerase and RCR proteins. The one or more reagents may be control nucleic acids, buffer solutions and/or saline solutions, including, for example, divalent metal ions, i.e., mg ²⁺ 、Mn ²⁺ 、Ca ²⁺ And/or Fe ²⁺ . The one or more reagents may produce single stranded nucleic acids.

The method of sequencing can produce a sequencing read that is at least about 70kb, at least about 80kb, at least about 90kb, at least about 100kb, at least about 110kb, at least about 120kb, at least about 130kb, at least about 140kb, at least about 150kb, at least about 160kb, at least about 170kb, at least about 180kb, at least about 190kb, at least about 200kb or longer in length.

In one example, the method of sequencing can be performed on an electrowetting array. The nucleic acid may be deoxyribonucleic acid (DNA).

The method of sequencing can produce sequencing data of at least about 100 gigabytes (Gb). The method of sequencing can produce data of at least about 10 Gb. The method of sequencing can produce sequencing data of at least about 30 Gb. The method of sequencing can produce sequencing data of at least about 500 Gb. The method of sequencing can produce sequencing data of at least about 512 Gb. The method of sequencing can produce sequencing data that produces at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500Gb or greater.

The circularized nucleic acid sample may comprise a plurality of sequences comprising a target sequence. At least about 80% of the plurality of sequences may comprise the target sequence. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of the plurality of sequences may comprise the target amplification.

Methods of sequencing can provide high purity nucleic acid samples. The purity of a nucleic acid sample can be assessed by measuring absorbance. Absorbance readings may be read on a spectrophotometer. A method of measuring purity may include providing a sample comprising at least one molecule; measuring absorbance of the sample at 260 nanometers (nm); measuring absorbance of the sample at 280 nm; and provides a ratio of absorbance at 260nm to absorbance at 280nm (A260/A280 ratio). The sequencing method may provide at least one sequencing read having an a260/a280 ratio of up to about 1.93. The sequencing method may provide at least one sequencing read having an a260/a280 ratio of up to about 1.84. The sequencing method can provide at least one sequencing read having an a260/a280 ratio of at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1 or less.

Next generation sequencing

The present invention provides methods of sequencing a nucleic acid sample. The method of sequencing may comprise Sequencing By Synthesis (SBS). Four different fluorescent-labeled deoxynucleotide triphosphates (dNTPs) and at least one polymerase may be provided. The at least one fluorescent dNTP may be integrated with the at least one nucleic acid molecule to be sequenced, followed by imaging. Fluorescent dye and terminator can be cleared from dntps. The sequencing of the at least one nucleic acid may continue in this manner. In one example, there are four images during each cycle, and each dNTP may emit a different intensity. In another example, two images may be taken during each cycle and the cyclic intensity may be plotted. Examples of SBS include, but are not limited to, cluster generation techniques by Illumina corporation (see "Technology Spotlight: illumina Sequencing Technology," which is incorporated herein by reference in its entirety).

The present disclosure provides methods of sequencing nucleic acid samples on an electrowetting array. The method may include combining a sample droplet with one or more reagent droplets; separating the sample droplet from the one or more reagent droplets; and combining the one or more reagent droplets with a second droplet. In one aspect, the one or more reagent droplets comprise at least one reagent for NGS. In one aspect, the one or more reagent droplets comprise at least one reagent for SBS. The agent may be part of a NovaSeq6000 Kit (see "NovaSeq 6000Reagent Kit", the entire contents of which are incorporated herein by reference), for example, illumina.

Next Generation Sequencing (NGS) library preparation and evaporation compensation

The evaporation compensation techniques described herein do not affect the reaction kinetics of NGS library preparation and are thus applicable to the various biological and chemical workflows described herein. In addition, multiple experiments can be performed with the same array for evaporative loss for each chemical/biological reaction, and a data set can be established. For example, the data set may be used to calculate the required compensation volume in order to keep the reaction volume within an error range of, for example, 20%, 10%, 5%, 1% or less. In reactions where there is a loss of volume, the compensation volume may be introduced in a timed manner (e.g., in an open loop without sensing and feedback). Alternatively, the data set may be input into a machine learning model to develop algorithms to learn how to estimate the compensation volume based on the characteristics of the response. The data set for input to the machine learning model may be generated by a sensor adjacent to the array or may be generated by a sensor external to the array. Likewise, improving the connected data set by simultaneous mixing and heating or improving the fragmented data set in response to active mixing on the array can be used to optimize performance of NGS sample preparation workflow using machine learning algorithms.

Polymerase Chain Reaction (PCR), clearance and quantitative PCR (qPCR)

Nucleic acid molecules can be amplified by thermal cycling based Polymerase Chain Reaction (PCR) on the arrays described herein. The fixed area of the array may be heated or cooled. Alternatively, different areas on the array may be heated or cooled to different temperatures or temperature ranges. For example, one or more droplets containing PCR reagents and sample may be shuttled back and forth between different areas of the array to perform PCR. A sensor (e.g., a fluorescence camera) may be used to illuminate and record the signal (e.g., fluorescence) of the droplets on the array. The detection may be performed in real time, providing qPCR functionality. For example, during qPCR operations, the signal may be read by monitoring dsDNA binding dyes (e.g., SYBR) or fluorescent probes (e.g., taqMan). During each PCR cycle, the signal may increase as the newly generated PCR product accumulates. For qPCR, an aliquot in a droplet may be used (e.g., droplet volume may be on the order of pL-mL). By monitoring qPCR in the aliquots in real time, the performance of the main sample can be inferred and the required amount of amplification can be adjusted accordingly. PCR and qPCR operations on an array or multiple arrays can be multiplexed to track various amplicons (e.g., genes, markers of interest, NGS libraries, etc.) in parallel. PCR and qPCR can be used for quantification of NGS libraries, gene expression, or target detection (e.g., diagnosis), for example.

Nano liter NGS

The amount of input material and reagents may be scaled down to nanoliter or picoliter sized reaction volumes (e.g., droplets) on the arrays described herein. The reagent concentration may be kept constant (e.g., for accurate reaction stoichiometry). The initial concentration and the final concentration of the reagent may not be kept constant (e.g., increased or decreased), for example, in order to optimize the reaction efficiency in nanoliter or picoliter-sized reaction volumes.

Nanoliter or picoliter sized droplets on the open face of an array (e.g., EWOD array or DEP array) or solid support (e.g., glass) can contact a much smaller array area than droplets sandwiched between two plates. Due to the smaller area occupancy, a large number of droplets (e.g., thousands of nanoliters and millions of picoliters) can fill in a small footprint of the array (e.g., the size of a standard SBS aperture plate). For example, on an open array with a smooth surface and no interfacial forces from the second surface (e.g., from the second plate), nano-sized droplets may be delivered and forces (e.g., electrodynamic forces from EWOD) applied for mixing. Furthermore, the droplets may be heated, cooled, placed in a magnetic field, or any combination thereof, for example. The actuation of nanoliter or picoliter sized droplets may be accomplished on electrodes of a size comparable to the droplet contact area (e.g., 0.00001 millimeters (mm), 0.0001mm, 0.001mm, 0.01mm, 0.1mm, 1mm, 10mm, 100mm, 1,000mm, or more). Alternatively, a set of successive electrodes surrounding nanoliter or picoliter sized droplets may be activated simultaneously to generate sufficient electrodynamic force to transport the droplets. The reaction volumes and electrode dimensions of this scale may provide reactions of at least about 1, 10, 100, 1,000, 10,000, 100,000, 1,000,000 or more in parallel (e.g., enabling nanoliter or picoliter scale high throughput applications). The process of scaling down the reaction, electrodes, input materials, reagents, or any combination thereof may be automated using software simulation.

Enzyme catalyzed biopolymer synthesis

Biopolymers (e.g., polynucleotides and polypeptides) can be synthesized on an array by dispensing and moving reagents sequentially, in parallel, or by a combination thereof. The reagent may include, for example, nucleoside triphosphates, nucleotides, enzymes, buffers, beads, deblocking agents, water, salts, or any combination thereof. For example, polynucleotide (e.g., DNA) synthesis may be performed directly on the surface of an array by functionalizing specific locations on the array. For example, the functionalized site may serve as a reaction site. DNA synthesis can also be performed on beads contained in droplets manipulated by an array (e.g., EWOD). DNA synthesis can be performed on an array in milliliter, microliter, nanoliter, picoliter, or femtoliter scale volumes. The DNA fragments can be assembled directly into longer fragments on the array by methods such as Gibson assembly. The combined combination of droplets can be used, for example, to generate a diversity of DNA fragments. The quality of the assembled DNA fragments can be assessed by preparing sequencing libraries on an array for downstream sequencing, e.g., sequencing based on illumina or Oxford nanopore technology, etc.

The present disclosure provides systems and methods for synthesizing at least one biopolymer on an array. In some embodiments, the sample droplet is contacted with at least one reagent droplet. In some embodiments, the sample droplet and the at least one reagent droplet are contacted by a droplet operation. In some embodiments, the at least one reagent comprises a reagent for enzyme-catalyzed biopolymer synthesis. Reagents and materials used in the methods disclosed herein can be found, for example, in U.S. patent No. 10,870,872, the entire contents of which are incorporated herein by reference. In some embodiments, a biosynthetic product is produced. In some aspects of the disclosure, the synthesized biological product is a polynucleotide. In other aspects of the disclosure, the synthesized biological product is a polypeptide.

Another aspect of the present disclosure is a method of producing a biopolymer, comprising: providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent; moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet, and (ii) form a combined droplet comprising the first reagent and the second reagent; and forming at least a portion of the biopolymer using at least (i) the first reagent and (ii) the second reagent in the combined droplet, wherein (b) - (c) are performed in 10 minutes or less. In some embodiments, the biopolymer is a polynucleotide. In some embodiments, the biopolymer is a polypeptide. In some embodiments, wherein the polynucleotide comprises about 10 to about 250 bases. In some embodiments, wherein the polynucleotide comprises about 260 to about 1kb. In some embodiments, the polynucleotide comprises about 1kb to about 10,000kb. In some embodiments, vibration is applied to the synthetic droplets during (b), (c), or both. In some embodiments, the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets. In some embodiments, vibration is applied to the one or more cleaning steps. In some embodiments, the surface is a dielectric. In some embodiments, the surface includes a dielectric layer disposed over the one or more electrodes. In some embodiments, the surface is a surface of a polymer film. In some embodiments, the surface comprises one or more oligonucleotides bound to the surface. In some embodiments, the surface is a surface of a lubricating liquid layer. In some embodiments, the plurality of droplets includes a third droplet comprising a third reagent. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof, comprises one or more functionalized beads. In some embodiments, the functionalized bead comprises one or more oligonucleotides immobilized thereto. In some embodiments, vibration is applied to the first droplet, the second droplet, the third droplet, the wash droplet, or a mixture thereof. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer. In some embodiments, the biomonomer is an amino acid. In some embodiments, the biomonomer is a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises one or more functionalized discs. In some embodiments, the functionalized disc comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof, comprises an enzyme that mediates synthesis or polymerization. In some embodiments, the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 20 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 15 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 10 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 1 minute or less. In some embodiments, the combined droplets are temperature controlled. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is exposed to light. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps). In some embodiments, the deoxynucleoside triphosphates can have a protecting group. In some embodiments, the protecting group may be removed in the reaction. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side. In some embodiments, the first, second, third, or combined droplet has a volume between 1 nanoliter (1 nL) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 200 microliters (200 μl). In some embodiments, the method further comprises attaching the biopolymer to a second biopolymer. In some embodiments, the second biopolymer is produced using any of the methods disclosed herein.

Another aspect of the present disclosure provides a method of producing a biopolymer, comprising: providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent; moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet, and (ii) form a combined droplet comprising the first reagent and the second reagent; and using at least (i) the first reagent and (ii) the second reagent in the combined droplets to form at least a portion of the biopolymer, wherein vibration is applied to (b), (c), or both. In some embodiments, the biopolymer is a polynucleotide. In some embodiments, the biopolymer is a polypeptide. In some embodiments, the polynucleotide comprises 2 to 10,000,000 nucleic acid molecules. In some embodiments, the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets. In some embodiments, vibration is applied to the one or more cleaning steps. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 30 minutes or less. In some embodiments, the surface is a dielectric. In some embodiments, the surface includes a dielectric layer disposed over the one or more electrodes. In some embodiments, the surface is a surface of a polymer film. In some embodiments, the surface comprises one or more oligonucleotides bound to the surface. In some embodiments, the surface is a surface of a lubricating liquid layer. In some embodiments, the plurality of droplets includes a third droplet comprising a third reagent. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof, comprises one or more functionalized beads. In some embodiments, the functionalized bead comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer. In some embodiments, the biomonomer is an amino acid. In some embodiments, the biomonomer is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the first agent comprises one or more functionalized discs. In some embodiments, the functionalized disc comprises one or more oligonucleotides immobilized thereto. In some embodiments, the first droplet, the second droplet, the third droplet, or both comprise an enzyme that mediates synthesis or polymerization. In some embodiments, the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family.

In some embodiments, the combined droplets are heated. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is exposed to light. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps). In some embodiments, the deoxynucleoside triphosphates can have a protecting group. In some embodiments, the protecting group may be removed in the reaction. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side. In some embodiments, the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 nanoliter (1 nL) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 500 microliters (500 μl). In some embodiments, the first, second, third, or combined droplet has a volume between 1 microliter (1 μl) and 200 microliters (200 μl).

In some aspects of the disclosure, the biopolymer is attached to a second biopolymer. In some embodiments, the second biopolymer is produced using any of the methods described in the present disclosure. The reservoir for storing the reagent (e.g., nucleoside triphosphates, magnetic beads, enzymes, salts, water, cleavage or deblocking agents) can be integrated on the surface of the array, integrated over the array, or dispensed from an external reservoir using the dispensing methods described herein.

The biopolymer may be a polynucleotide. Polynucleotides may be at least 10, 100, 1,000, 10,000, 100,000, 1,000,000 or more base pairs long. In some embodiments, the polynucleotide is about 1 base in length. In some embodiments, the polynucleotide is about 1 base to about 750 bases in length. In some embodiments of the present invention, in some embodiments, the polynucleotide is about 1 base to about 10 bases, about 1 base to about 20 bases, about 1 base to about 50 bases, about 1 base to about 100 bases, about 1 base to about 150 bases, about 1 base to about 200 bases, about 1 base to about 250 bases, about 1 base to about 500 bases, about 1 base to about 750 bases, about 1 base to about 1 base, about 10 bases to about 20 bases, about 10 bases to about 50 bases, about 10 bases to about 100 bases, about 10 bases to about 150 bases, about 10 bases to about 200 bases, about 10 bases to about 250 bases, about 10 bases to about 500 bases, about 10 bases to about 750 bases, about 10 bases to about 1 base, about 20 bases to about 50 bases, about 20 bases to about 100 bases, about about 20 bases to about 150 bases, about 20 bases to about 200 bases, about 20 bases to about 250 bases, about 20 bases to about 500 bases, about 20 bases to about 750 bases, about 20 bases to about 1 base, about 50 bases to about 100 bases, about 50 bases to about 150 bases, about 50 bases to about 200 bases, about 50 bases to about 250 bases, about 50 bases to about 500 bases, about 50 bases to about 750 bases, about 50 bases to about 1 base, about 100 bases to about 150 bases, about 100 bases to about 200 bases, about 100 bases to about 250 bases, about 100 bases to about 500 bases, about 100 bases to about 750 bases, about 100 bases to about 1 base, about 150 bases to about 200 bases, about 150 bases to about 250 bases, about 100 bases to about 1 base, about, about 150 bases to about 500 bases, about 150 bases to about 750 bases, about 150 bases to about 1 base, about 200 bases to about 250 bases, about 200 bases to about 500 bases, about 200 bases to about 750 bases, about 200 bases to about 1 base, about 250 bases to about 500 bases, about 250 bases to about 750 bases, about 250 bases to about 1 base, about 500 bases to about 750 bases, about 500 bases to about 1 base, or about 750 bases to about 1 base. In some embodiments, the polynucleotide is about 1 base, about 10 bases, about 20 bases, about 50 bases, about 100 bases, about 150 bases, about 200 bases, about 250 bases, about 500 bases, about 750 bases, or about 1 base in length. In some embodiments, the polynucleotide is at least about 1 base, about 10 bases, about 20 bases, about 50 bases, about 100 bases, about 150 bases, about 200 bases, about 250 bases, about 500 bases, or about 750 bases in length. In some embodiments, the polynucleotide is up to about 10 bases, about 20 bases, about 50 bases, about 100 bases, about 150 bases, about 200 bases, about 250 bases, about 500 bases, about 750 bases, or about 1 base in length.

In some embodiments, the polynucleotide is about 1 kilobase (kb) to about 250 kilobases (kb) in length. In some embodiments, the polynucleotide is about 1 kilobase (kb) to about 2 kilobases (kb), about 1 kilobase (kb) to about 3 kilobases (kb), about 1 kilobase (kb) to about 4 kilobases (kb), about 1 kilobase (kb) to about 5 kilobases (kb), about 1 kilobase (kb) to about 10 kilobases (kb), about 1 kilobase (kb) to about 20 kilobases (kb), about 1 kilobase (kb) to about 50 kilobases (kb), about 1 kilobase (kb) to about 100 kilobases (kb), about 1 kilobase (kb) to about 150 kilobases (kb), about 1 kilobase (kb) to about 200 kilobases (kb), about 1 kilobase (kb) to about 250 kilobases (kb), about 2 kilobases (kb) to about 3 kilobases (kb), about 2 kilobases (kb) to about 4 kilobases (kb), about 2 kilobases (kb) to about 5 kilobases (kb) to about 2 kilobases (kb), about 2 kilobases (kb) to about 20 kb (kb), about 2 kilobases (kb) to about 150 kilobases (kb), about 2 kilobases (kb) to about 200 kilobases (kb), about 2 kilobases (kb) to about 250 kilobases (kb), about 3 kilobases (kb) to about 4 kilobases (kb), about 3 kilobases (kb) to about 5 kilobases (kb), about 3 kilobases (kb) to about 10 kilobases (kb), about 3 kilobases (kb) to about 20 kilobases (kb), about 3 kilobases (kb) to about 50 kilobases (kb), about 3 kilobases (kb) to about 100 kilobases (kb), about 3 kilobases (kb) to about 150 kilobases (kb), about 3 kilobases (kb) to about 200 kilobases (kb), about 3 kilobases (kb) to about 250 kilobases (kb), about 4 kilobases (kb) to about 5 kilobases (kb), about 4 kilobases (kb) to about 10 kilobases (kb), about 4 kilobases (kb) to about 20 kilobases (kb), about 4 kilobases (kb) to about 150 kilobases (kb), about 4 kilobases (kb) to about 20 kilobases (kb) to about 100 kilobases (kb), about 4 kilobases (kb) to about 200 kilobases (kb), about 4 kilobases (kb) to about 250 kilobases (kb), about 5 kilobases (kb) to about 10 kilobases (kb), about 5 kilobases (kb) to about 20 kilobases (kb), about 5 kilobases (kb) to about 50 kilobases (kb), about 5 kilobases (kb) to about 100 kilobases (kb), about 5 kilobases (kb) to about 150 kilobases (kb), about 5 kilobases (kb) to about 200 kilobases (kb), about 5 kilobases (kb) to about 250 kilobases (kb), about 10 kilobases (kb) to about 20 kilobases (kb), about 10 kilobases (kb) to about 50 kilobases (kb), about 10 kilobases (kb) to about 100 kilobases (kb), about 10 kilobases (kb) to about 150 kilobases (kb), about 10 kilobases (kb) to about 100 kilobases (kb), about 200 kilobases (kb) to about 20 kilobases (kb), about 250 kilobases (kb) to about 20 kilobases (kb) to about 250 kilobases (kb), about 20 kilobases (kb) to about 200 kilobases (kb), about 20 kilobases (kb) to about 250 kilobases (kb), about 50 kilobases (kb) to about 100 kilobases (kb), about 50 kilobases (kb) to about 150 kilobases (kb), about 50 kilobases (kb) to about 200 kilobases (kb), about 50 kilobases (kb) to about 250 kilobases (kb), about 100 kilobases (kb) to about 150 kilobases (kb), about 100 kilobases (kb) to about 200 kilobases (kb), about 100 kilobases (kb) to about 250 kilobases (kb), about 150 kilobases (kb) to about 200 kilobases (kb), about 150 kilobases (kb) to about 250 kilobases (kb), or about 200 kilobases (kb) to about 250 kilobases (kb). In some embodiments, the polynucleotide is about 1 kilobase (kb), about 2 kilobases (kb), about 3 kilobases (kb), about 4 kilobases (kb), about 5 kilobases (kb), about 10 kilobases (kb), about 20 kilobases (kb), about 50 kilobases (kb), about 100 kilobases (kb), about 150 kilobases (kb), about 200 kilobases (kb), or about 250 kilobases (kb) in length. In some embodiments, the polynucleotide is at least about 1 kilobase (kb), about 2 kilobases (kb), about 3 kilobases (kb), about 4 kilobases (kb), about 5 kilobases (kb), about 10 kilobases (kb), about 20 kilobases (kb), about 50 kilobases (kb), about 100 kilobases (kb), about 150 kilobases (kb), or about 200 kilobases (kb) in length. In some embodiments, the polynucleotide is up to about 2 kilobases (kb), about 3 kilobases (kb), about 4 kilobases (kb), about 5 kilobases (kb), about 10 kilobases (kb), about 20 kilobases (kb), about 50 kilobases (kb), about 100 kilobases (kb), about 150 kilobases (kb), about 200 kilobases (kb), or about 250 kilobases (kb) in length.

In some embodiments, the polynucleotide is about 250 kilobases (kb) to about 10,000 kilobases (kb) in length. In some embodiments, the polynucleotide is about 250 kilobases (kb) to about 500 kilobases (kb), about 250 kilobases (kb) to about 750 kilobases (kb), about 250 kilobases (kb) to about 1,000 kilobases (kb), about 250 kilobases (kb) to about 2,000 kilobases (kb), about 250 kilobases (kb) to about 3,000 kilobases (kb), about 250 kilobases (kb) to about 4,000 kilobases (kb), about 250 kilobases (kb) to about 5,000 kilobases (kb), about 250 kilobases (kb) to about 10,000 kilobases (kb), about 500 kilobases (kb) to about 750 kilobases (kb), about 500 kilobases (kb) to about 1,000 kilobases (kb), about 500 kilobases (kb) to about 2,000 kilobases (kb), about 500 kilobases (kb) to about 3,000 bases (kb), about 250 kilobases (kb) to about 4,000 kilobases (kb), about 250 kilobases (kb) to about 5,000 kilobases (kb) to about 750 kilobases (kb), about 500 kilobases (kb) to about 5,000 kilobases (kb) to about 750 kb (kb) to about 5,000 kilobases (kb) to about 5,000 kb (kb) About 750 kilobases (kb) to about 4,000 kilobases (kb), about 750 kilobases (kb) to about 5,000 kilobases (kb), about 750 kilobases (kb) to about 10,000 kilobases (kb), about 1,000 kilobases (kb) to about 2,000 kilobases (kb), about 1,000 kilobases (kb) to about 3,000 kilobases (kb), about 1,000 kilobases (kb) to about 4,000 kilobases (kb), about 1,000 kilobases (kb) to about 5,000 kilobases (kb), about 1,000 kilobases (kb) to about 10,000 kilobases (kb), about 2,000 kilobases (kb) to about 3,000 kilobases (kb), about 2,000 kilobases (kb) to about 5,000 bases (kb), about 1,000 kilobases (kb) to about 5,000 kilobases (kb), about 1,000 kilobases (kb) to about 10,000 kilobases (kb) to about 3,000 kilobases (kb), about 5,000 kilobases (kb) to about 3,000 kilobases (kb) to about 5,000 kb). In some embodiments, the polynucleotide is about 250 kilobases (kb), about 500 kilobases (kb), about 750 kilobases (kb), about 1,000 kilobases (kb), about 2,000 kilobases (kb), about 3,000 kilobases (kb), about 4,000 kilobases (kb), about 5,000 kilobases (kb), or about 10,000 kilobases (kb) in length. In some embodiments, the polynucleotide is at least about 250 kilobases (kb), about 500 kilobases (kb), about 750 kilobases (kb), about 1,000 kilobases (kb), about 2,000 kilobases (kb), about 3,000 kilobases (kb), about 4,000 kilobases (kb), or about 5,000 kilobases (kb) in length. In some embodiments, the polynucleotide is up to about 500 kilobases (kb), about 750 kilobases (kb), about 1,000 kilobases (kb), about 2,000 kilobases (kb), about 3,000 kilobases (kb), about 4,000 kilobases (kb), about 5,000 kilobases (kb), or about 10,000 kilobases (kb) in length.

In some aspects of the disclosure, the polynucleotide comprises from about 2 to about 10,000,000 nucleic acid molecules. In some embodiments, the polynucleotide comprises from about 1 nucleic acid to about 1,000 nucleic acids. In some embodiments of the present invention, in some embodiments, polynucleotides comprise from about 1 nucleic acid to about 2 nucleic acids, from about 1 nucleic acid to about 5 nucleic acids, from about 1 nucleic acid to about 10 nucleic acids, from about 1 nucleic acid to about 25 nucleic acids, from about 1 nucleic acid to about 50 nucleic acids, from about 1 nucleic acid to about 100 nucleic acids, from about 1 nucleic acid to about 250 nucleic acids, from about 1 nucleic acid to about 500 nucleic acids, from about 1 nucleic acid to about 750 nucleic acids, from about 1 nucleic acid to about 1,000 nucleic acids, from about 2 nucleic acids to about 5 nucleic acids, from about 2 nucleic acids to about 10 nucleic acids, from about 2 nucleic acids to about 25 nucleic acids, from about 2 nucleic acids to about 50 nucleic acids, from about 2 nucleic acids to about 100 nucleic acids, from about 2 nucleic acids to about 250 nucleic acids, from about 2 nucleic acids to about 500 nucleic acids, from about 2 nucleic acids to about 750 nucleic acids, from about 2 nucleic acids to about 1,000 nucleic acids, from about 5 nucleic acids to about 10 nucleic acids, from about 5 nucleic acids to about 25 nucleic acids, from about 2 nucleic acids about 5 to about 50 nucleic acids, about 5 to about 100 nucleic acids, about 5 to about 250 nucleic acids, about 5 to about 500 nucleic acids, about 5 to about 750 nucleic acids, about 5 to about 1,000 nucleic acids, about 10 to about 25 nucleic acids, about 10 to about 50 nucleic acids, about 10 to about 100 nucleic acids, about 10 to about 250 nucleic acids, about 10 to about 500 nucleic acids, about 10 to about 750 nucleic acids, about 10 to about 1,000 nucleic acids, about 25 to about 50 nucleic acids, about 25 to about 100 nucleic acids, about 25 to about 250 nucleic acids, about 25 to about 500 nucleic acids, about 25 to about 750 nucleic acids, about 25 to about 1,000 nucleic acids, about 50 to about 100 nucleic acids, about 50 to about 250 nucleic acids, about, about 50 to about 500 nucleic acids, about 50 to about 750 nucleic acids, about 50 to about 1,000 nucleic acids, about 100 to about 250 nucleic acids, about 100 to about 500 nucleic acids, about 100 to about 750 nucleic acids, about 100 to about 1,000 nucleic acids, about 250 to about 500 nucleic acids, about 250 to about 750 nucleic acids, about 250 to about 1,000 nucleic acids, about 500 to about 750 nucleic acids, about 500 to about 1,000 nucleic acids, or about 750 to about 1,000 nucleic acids. In some embodiments, the polynucleotide comprises about 1 nucleic acid, about 2 nucleic acids, about 5 nucleic acids, about 10 nucleic acids, about 25 nucleic acids, about 50 nucleic acids, about 100 nucleic acids, about 250 nucleic acids, about 500 nucleic acids, about 750 nucleic acids, or about 1,000 nucleic acids. In some embodiments, the polynucleotide comprises at least about 1 nucleic acid, about 2 nucleic acids, about 5 nucleic acids, about 10 nucleic acids, about 25 nucleic acids, about 50 nucleic acids, about 100 nucleic acids, about 250 nucleic acids, about 500 nucleic acids, or about 750 nucleic acids. In some embodiments, the polynucleotide comprises up to about 2 nucleic acids, about 5 nucleic acids, about 10 nucleic acids, about 25 nucleic acids, about 50 nucleic acids, about 100 nucleic acids, about 250 nucleic acids, about 500 nucleic acids, about 750 nucleic acids, or about 1,000 nucleic acids.

In some embodiments, the polynucleotide comprises from about 1,000 nucleic acids to about 100,000 nucleic acids. In some embodiments, the polynucleotide comprises from about 1,000 to about 2,000 nucleic acids, from about 1,000 to about 5,000 nucleic acids, from about 1,000 to about 7,500 nucleic acids, from about 1,000 to about 10,000 nucleic acids, from about 1,000 to about 20,000 nucleic acids, from about 1,000 to about 50,000 nucleic acids, from about 1,000 to about 75,000 nucleic acids, from about 1,000 to about 5,000 nucleic acids, from about 2,000 to about 7,500 nucleic acids, from about 2,000 to about 10,000 nucleic acids, from about 2,000 to about 20,000 nucleic acids, from about 2,000 to about 50,000 nucleic acids, from about 2,000 to about 75,000 nucleic acids, from about 2,000 to about 100,000 nucleic acids, from about 5,000 to about 5,000, from about 5,000 to about 10,000, from about 5,000 to about 50,000, from about 10,000 to about 50,000, from about 5,000 to about 10,000, from about 10,000 to about 50,000, from about 10,000, from about 5,000 to about 50,000, from about 10,000 to about 50,000, from about 10,000 to about 50,000. In some embodiments, the polynucleotide comprises about 1,000 nucleic acids, about 2,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 20,000 nucleic acids, about 50,000 nucleic acids, about 75,000 nucleic acids, or about 100,000 nucleic acids. In some embodiments, the polynucleotide comprises at least about 1,000 nucleic acids, about 2,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 20,000 nucleic acids, about 50,000 nucleic acids, or about 75,000 nucleic acids. In some embodiments, the polynucleotide comprises up to about 2,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 20,000 nucleic acids, about 50,000 nucleic acids, about 75,000 nucleic acids, or about 100,000 nucleic acids.

In some embodiments, the polynucleotide comprises from about 100,000 nucleic acids to about 10,000,000 nucleic acids. In some embodiments, the polynucleotide comprises from about 100,000 to about 200,000 nucleic acids, from about 100,000 to about 750,000 nucleic acids, from about 100,000 to about 1,000,000 nucleic acids, from about 100,000 to about 2,000,000 nucleic acids, from about 100,000 to about 5,000,000 nucleic acids, from about 100,000 to about 7,500,000 nucleic acids, from about 100,000 to about 10,000,000 nucleic acids, from about 200,000 to about 750,000 nucleic acids, from about 200,000 to about 1,000,000 nucleic acids, from about 200,000 to about 2,000,000 nucleic acids, from about 200,000 to about 5,000,000 nucleic acids, from about 200,000 to about 7,500,000 nucleic acids, from about 200,000 to about 10,000 nucleic acids, from about 750,000 to about 750,000,000 nucleic acids, from about 5,000 to about 5,000,000,000, from about 5,000 to about 7,000,000,000, from about 5,000,000 to about 7,000,000,000, from about 5,000,000,000 to about 7,000,000,000, from about 5,000,000,000,000 to about 7,000,000,000,000,000, from about 5,000,000,000,000,000 to about 500,000,000,000,000,000,000,000, from about 7,000,000,000,000,000,000,000,000,. In some embodiments, the polynucleotide comprises about 100,000 nucleic acids, about 200,000 nucleic acids, about 750,000 nucleic acids, about 1,000,000 nucleic acids, about 2,000,000 nucleic acids, about 5,000,000 nucleic acids, about 7,500,000 nucleic acids, or about 10,000,000 nucleic acids. In some embodiments, the polynucleotide comprises at least about 100,000 nucleic acids, about 200,000 nucleic acids, about 750,000 nucleic acids, about 1,000,000 nucleic acids, about 2,000,000 nucleic acids, about 5,000,000 nucleic acids, or about 7,500,000 nucleic acids. In some embodiments, the polynucleotide comprises up to about 200,000 nucleic acids, about 750,000 nucleic acids, about 1,000,000 nucleic acids, about 2,000,000 nucleic acids, about 5,000,000 nucleic acids, about 7,500,000 nucleic acids, or about 10,000,000 nucleic acids.

In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 20 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 15 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated within the combined droplets in 10 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is generated in about 5 minutes or less to about 20 minutes or less. In some embodiments of the present invention, in some embodiments, at least one nucleic acid molecule of the polynucleotide is within a range of about 20 minutes or less to about 19 minutes or less, within a range of about 20 minutes or less to about 18 minutes or less, within a range of about 20 minutes or less to about 17 minutes or less, within a range of about 20 minutes or less to about 16 minutes or less, within a range of about 20 minutes or less to about 15 minutes or less, within a range of about 20 minutes or less to about 14 minutes or less, within a range of about 20 minutes or less to about 13 minutes or less, within a range of about 20 minutes or less to about 12 minutes or less, within a range of about 20 minutes or less to about 11 minutes or less, within a range of about 20 minutes or less to about 10 minutes or less, within a range of about 20 minutes or less to about 5 minutes or less, within a range of about 20 minutes or less about 19 minutes or less to about 18 minutes or less, about 19 minutes or less to about 17 minutes or less, about 19 minutes or less to about 16 minutes or less, about 19 minutes or less to about 15 minutes or less, about 19 minutes or less to about 14 minutes or less, about 19 minutes or less to about 13 minutes or less, about 19 minutes or less to about 12 minutes or less, about 19 minutes or less to about 11 minutes or less, about 19 minutes or less to about 10 minutes or less, about 19 minutes or less to about 5 minutes or less, about 18 minutes or less to about 17 minutes or less, about 19 minutes or less, about 5 minutes or less, about 18 minutes or less, about 18 minutes or less to about 16 minutes or less, about 18 minutes or less to about 15 minutes or less, about 18 minutes or less to about 14 minutes or less, about 18 minutes or less to about 13 minutes or less, about 18 minutes or less to about 12 minutes or less, about 18 minutes or less to about 11 minutes or less, about 18 minutes or less to about 10 minutes or less, about 18 minutes or less to about 5 minutes or less, about 17 minutes or less to about 16 minutes or less, about 17 minutes or less to about 15 minutes or less, about 17 minutes or less to about 14 minutes or less, about 17 minutes or less to about 13 minutes or less about 17 minutes or less to about 12 minutes or less, about 17 minutes or less to about 11 minutes or less, about 17 minutes or less to about 10 minutes or less, about 17 minutes or less to about 5 minutes or less, about 16 minutes or less to about 15 minutes or less, about 16 minutes or less to about 14 minutes or less, about 16 minutes or less to about 13 minutes or less, about 16 minutes or less to about 12 minutes or less, about 16 minutes or less to about 11 minutes or less, about 16 minutes or less to about 10 minutes or less, about 16 minutes or less to about 5 minutes or less, about 15 minutes or less to about 14 minutes or less, about 16 minutes or less to about 11 minutes or less, about 15 minutes or less to about 13 minutes or less, about 15 minutes or less to about 12 minutes or less, about 15 minutes or less to about 11 minutes or less, about 15 minutes or less to about 10 minutes or less, about 15 minutes or less to about 5 minutes or less, about 14 minutes or less to about 13 minutes or less, about 14 minutes or less to about 12 minutes or less, about 14 minutes or less to about 11 minutes or less, about 14 minutes or less to about 10 minutes or less, about 14 minutes or less to about 5 minutes or less, about 13 minutes or less to about 12 minutes or less, about 13 minutes or less to about 11 minutes or less, about 13 minutes or less to about 10 minutes or less, about 13 minutes or less to about 5 minutes or less, about 12 minutes or less to about 11 minutes or less, about 11 minutes or less to about 11 minutes or less, about 10 minutes or less, about 11 minutes or less to about 10 minutes or less, about 11 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is produced in about 20 minutes or less, about 19 minutes or less, about 18 minutes or less, about 17 minutes or less, about 16 minutes or less, about 15 minutes or less, about 14 minutes or less, about 13 minutes or less, about 12 minutes or less, about 11 minutes or less, about 10 minutes or less, or about 5 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is produced in at least about 20 minutes or less, about 19 minutes or less, about 18 minutes or less, about 17 minutes or less, about 16 minutes or less, about 15 minutes or less, about 14 minutes or less, about 13 minutes or less, about 12 minutes or less, about 11 minutes or less, or about 10 minutes or less. In some embodiments, at least one nucleic acid molecule of the polynucleotide is produced in up to about 19 minutes or less, about 18 minutes or less, about 17 minutes or less, about 16 minutes or less, about 15 minutes or less, about 14 minutes or less, about 13 minutes or less, about 12 minutes or less, about 11 minutes or less, about 10 minutes or less, or about 5 minutes or less.

1. 10, 100, 1,000, 10,000, 100,000, 1,000,000 or more reactions can be performed in parallel on a single array or multiple arrays. Droplets with polynucleotide (e.g., DNA) sequences can be purified and size selected using magnetic beads. Purified DNA sequences can be combined and assembled in a combinatorial fashion using DNA assembly techniques, such as Gibson assembly, and the like. The assembled polynucleotide (e.g., DNA) may contain errors. To correct errors, the assembled polynucleotide (e.g., DNA) may be treated with a mismatch binding protein or a mismatch cleaving protein (e.g., mutS, T4 endonuclease VII, or T7 endonuclease I).

Arrays for synthesizing DNA using enzyme-catalyzed methods may be stacked vertically or horizontally as described herein. The stack may be connected to a cloud server infrastructure. For example, when a user purchases a sequence of, for example, DNA, gene libraries, RNA, guide RNA, or other biopolymers, the user can interact with a dashboard on a computer directly connected to the cloud infrastructure. When submitting an input sequence for synthesis, a limited set of arrays may be instantiated as required. For example, the number of arrays may range from one to several billion. Once the array is instantiated, the entire synthesis process can run autonomously.

Sample quantification

Optically-based (e.g., fluorescence) detection of nucleic acids (e.g., DNA) on an array can be accomplished by using, for example, intercalating fluorescent dyes (e.g., SYBR Green). For fluorescence-based measurements, the sample may be positioned into the sample detection zone (5710) from another portion of the array. The sample detection zone may be an optically clear path (e.g., a transparent or surface aperture). An excitation light source, excitation filter, mirror, emission filter, detection sensor, or any combination thereof may be disposed below the sample to excite the light and return through an optically clear path.

For example, a size selection unit may precede the fluorescence-based detection zone. Size-based separation units can employ electrophoresis or capillary electrophoresis and perform separation based on the size of the nucleic acid fragments. The size-separated sample may pass through a detection zone, wherein a fluorescent signal profile of the sample may be indicative of a size profile of the sample. The total fluorescence of the sample can be used to quantify the concentration of total nucleic acid in the sample.

The nucleic acid sequencer may be a Maxam-Gilbert sequencer or a Sanger sequencer. The bioprotein channel may be a biological nanopore. The bioprotein channel may be a hemolysin or MspA porin. The solid state nanopore may be silicon nitride or graphene. The protein sequencer may be a mass spectrometer, a single molecule sequencer or an Edman degradation sequencer. Nucleic acid sequencing may include sequencing by synthesis, pyrosequencing, sequencing by hybridization, sequencing by ligation, sequencing by detection of ions released during DNA polymerization, single molecule sequencing, or any combination thereof. Single molecule sequencing may be nanopore sequencing. Single molecule sequencing may be Single Molecule Real Time (SMRT) sequencing.

In some embodiments, nucleic acid and/or protein sequencing/identification assays can be integrated into the EWOD systems, devices, and/or arrays described herein. In some embodiments, nanopore sensors can be integrated into EWOD systems, devices, and/or arrays described herein for biomolecule sensing and sequencing. In some embodiments, the EWOD systems, devices, and/or arrays and nanopore sensors described herein can all be fabricated in a single piece of silicon. In some embodiments, the EWOD systems, devices, and/or arrays described herein can be fabricated with standard electronics manufacturing practices (e.g., as described herein), and the nanopore sensor can be fabricated in a silicon process and mounted or coupled adjacent to the EWOD systems, devices, and/or arrays described herein. In some embodiments, the nanopore sensor may contain protein-based pores for sensing, or may also be entirely solid state.

In some embodiments, the nanopores may be integrated into the EWOD systems, devices, and/or arrays described herein on the same plane as the droplets. In some embodiments, the nanopore may be located above, below, or to the side of the electrowetting surface/array. The EWOD systems, devices, and/or arrays described herein can have pores through which droplets containing biological samples are transferred to nanopore sensors.

One embodiment of this aspect of the present disclosure is illustrated schematically in fig. 23.

The acoustic sensor can be a subsonic wave, an ultrasonic wave, or a combination thereof. The acoustic sensor may be coupled to the array by an acoustic coupling medium. The acoustic coupling medium may be solid or liquid. MEMS sensors may measure force, pressure, or temperature. The capillary tube as a liquid dispenser may be about 2 millimeters (mm) in diameter, 1.5mm in diameter, 1mm in diameter, 0.5mm in diameter, 0.25mm in diameter, or less. There may be 1,2, 3, 4, 5, 10, 50, 100 or more capillaries in an array. The wells that use gravity to dispense or transfer liquid can be treated with different materials to increase or decrease the hydrophobicity of the wells. There may be 1,2, 3, 4, 5, 10, 50, 100 or more wells in the array. The pores may be at least about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm, 1,100 μm, 1,200 μm, 1,300 μm, 1,400 μm, 1,500 μm, 1,600 μm, 1,700 μm, 1,800 μm, 1,900 μm, 2,000 μm or larger in diameter. The pores may be up to about 2,000 μm, 1,900 μm, 1,800 μm, 1,700 μm, 1,600 μm, 1,500 μm, 1,400 μm, 1,300 μm, 1,200 μm, 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm or less in diameter. The diameter of the pores may be 100 μm to 500 μm. The electrodes in the wells for dispensing or transferring liquid may utilize the electrowetting effect. These holes can be used for optical detection. The apertures may have the dimensions described herein. The pores for the interaction of liquids through the membrane may have a membrane of the materials described herein. These apertures may be used in any combination of liquid dispensing or transfer using electric fields, aerodynamic, optical detection, to allow liquid interaction through the membrane.

The array may interface with a liquid handling unit, which may direct a plurality of droplets adjacent to the array. The liquid handling unit may be selected from robotic liquid handling systems, acoustic liquid dispensers, syringe pumps, spray nozzles, microfluidic devices, needles, diaphragm-based pump dispensers, piezoelectric pumps, and other liquid dispensers. The robotic liquid handling system may be a stationary liquid dispensing platform or an electrically powered platform for mapping liquid dispensing. The robotic liquid handling system may have one or more gun heads for dispensing liquid. The acoustic liquid dispenser may dispense a liquid volume of less than 1 nanoliter (nL). The acoustic liquid dispenser may have about 1 to 1600 apertures for liquid storage. The syringe pump may be configured to process 1 to 10 or more syringes in parallel. The syringe pump may use a syringe having a volume of less than 1mL to 50mL or more. The spray nozzle may be a fixed nozzle or a disposable head nozzle. The spray nozzles may comprise an array of from about 1 nozzle to 10 nozzles or more. The ejection nozzles may be driven by piezo actuators or hot droplet generation. The microfluidic device may comprise an array of microfluidic channels from 1 channel to 1000 or more channels. Microfluidic devices may be used to initiate a reaction before a liquid is dispensed into a droplet. The needle size may range from less than 7 gauge to 24 gauge or more. The needles may comprise an array having a number of needles from 1 needle to 100 needles or more. The diaphragm pump may have a diaphragm made of rubber, thermoplastic, fluoropolymer, other plastic, or any combination thereof.

The array may be connected to a reagent storage unit, a sample storage unit, a plurality of reagent storage units, a plurality of sample storage units, or any combination thereof. The reagent storage unit, sample storage unit, plurality of reagent storage units, plurality of sample storage units, or any combination thereof may comprise at least one multi-well plate, test tube, bottle, reservoir, jet cartridge, plate, petri dish, or any combination thereof. The multi-well plate may include at least about 2, 6, 12, 24, 48, 96, 384, 1536, 3456, 9600, or more wells. The test tube may be selected from Eppendorf or Falcon tubes. The bottle may be made of glass, polycarbonate, polyethylene or other materials compatible with the substances that may be stored in the bottle. The bottle can have a capacity of greater than about 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL, 900mL, 1L, 2L, 3L, 4L, 5L, or more. These bottles are replicable. The reservoir may be a High Performance Liquid Chromatography (HPLC) solvent reservoir. The reservoir may be made of glass, polycarbonate, polyethylene, or other materials that are compatible with the substances that may be stored in the reservoir. The reservoir can have a capacity of greater than about 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL, 900mL, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, 50L, or more. The jetting cartridges may be commercially available, specially manufactured for arrays, or a combination thereof. The jetting cartridge may dispense the liquid thermally, piezoelectrically, or a combination thereof. The jetting cartridge may be refillable, disposable, or have refillable and disposable components. The spray cartridge may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different liquids. The plate may be a medium for cell growth. The medium used for cell growth may be agar. Agar may contain nutrients for promoting cell growth. The nutrients used to promote cell growth may be blood, blood-derived nutrients, sugars, other essential nutrients, or any combination thereof. The petri dish may comprise a plate. The dish may be bare. The petri dish may be made of glass, plastic or a combination thereof. The petri dish may be a replica organism detection and counting (RODAC) plate. The plurality of wells of the multi-well plate may be heat conducting wells, electron receiving wells, or a combination thereof. The reagent or sample may be manipulated inside or outside the well by an electric field, magnetic field, acoustic wave, heat, pressure, vibration, liquid handling unit, or a combination thereof.

The array may include a coating. The coating may be a hydrophobic coating. The coating may be a hydrophilic coating. The coating may include a hydrophobic coating and a hydrophilic coating. The coating may be cleaned by washing. The coating may reduce evaporation. The coating may reduce evaporation by 10% to 100%. The coating may reduce evaporation by 50% to 100%. The coating may reduce biofouling. The coating may reduce biofouling by 10% to 100%. The coating may be resistant to biofouling. The coating may be resistant to biofouling. The hydrophobic coating may be a fluoropolymer, polyethylene or polystyrene. The hydrophobic coating may also be a surface modified with molecules such as fatty acids, polyaromatic compounds, and the like. For example, oleic acid may bind to a surface, forming a carbon chain that increases the hydrophobicity of the surface. The hydrophilic coating may be a hydrophilic polymer such as polyvinyl alcohol, polyethylene glycol, and the like. The coating comprising both a hydrophobic coating and a hydrophilic coating may be a combination of the hydrophilic and hydrophobic polymers described above, or may be a polymer having both hydrophilicity and hydrophobicity, such as a copolymer.

The coating can be easily cleaned by washing. Such a coating may be smooth for the samples placed thereon to facilitate easy removal of the samples. The droplet may include a coating to prevent or reduce evaporation of material from within the droplet to the environment outside the droplet, from the environment to the inside of the droplet, or any combination thereof. Such a coating may reduce evaporation of the contents inside the droplet by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. The coating may be a polymeric coating (e.g., polyethylene glycol). The coating may be formed as a skin surrounding the droplets. For example, the coating may be generated by contacting the droplets with a fluid comprising a polymeric material (e.g., a polymer or a polymer precursor). When the polymeric material comes into contact with water droplets, diffusion of the fluid into the water induces polymerization or cross-linking.

The coating may reduce the accumulation of biofouling or undesirable biological species as being biocidal or non-toxic. An example of a biocidal coating may be a coating containing molecules that are toxic to biological systems, such as tributyltin or other biocidal agents. Examples of non-toxic coatings may include coatings that reduce the attachment of biological species, such as fluoropolymers or polydimethylsiloxanes. Such a coating may act as an anti-biofouling.

The coefficient of variation may be less than 15%, 10%, 5% or 1%. For example, a variation coefficient of the droplet size of 1% means that the standard deviation of the droplet size variation divided by the average decrease of the droplet size is 1% for the same series of processes performed on a plurality of droplets.

The processing of the plurality of biological samples may include nucleic acid sequencing. Nucleic acid sequencing may include Polymerase Chain Reaction (PCR). The PCR may include a high multiplex PCR, a quantitative PCR, a drop digital PCR, a reverse transcriptase PCR, or any combination thereof. The highly multiplex PCR may be a single template or a multi-template PCR reaction. Quantitative PCR can use a variety of labels to display PCR products in real time, such as SYBR Green or TaqMan probes. Drop digital PCR can use initial drops from less than 1 microliter to greater than 50 microliters and can separate these drops into greater than 10,000 drops by oil-water emulsion techniques. Reverse transcriptase PCR may be one step or two steps (i.e., only one droplet or multiple droplets may be required to complete). Reverse transcriptase PCR can use fluorescence measurements to endpoint determination or real-time quantification of products.

The processing of the plurality of biological samples may include sample preparation for genomic sequencing. Preparation for genomic sequencing may include removal of DNA from the host cell, cell-free DNA, or any combination thereof. Preparation for genomic sequencing may include amplification to provide sufficient DNA for sequencing. Preparation for genomic sequencing may utilize enzymatic fragmentation of DNA, mechanical fragmentation of DNA, or any combination thereof.

The processing of multiple biological samples may include combinatorial assembly of genes. The combinatorial assembly of genes may include Gibson assembly, restriction enzyme cloning, gBlock fragment assembly (IDT), bioBricks assembly, NEBuilder HiFi DNA assembly, golden Gate assembly, site-directed mutagenesis, sequence and Ligase Independent Cloning (SLIC), cyclic Polymerase Extension Cloning (CPEC) and seamless ligation cloning extract (SLiCE), topoisomerase mediated ligation, homologous recombination, gateway cloning, geneArt gene synthesis, or any combination thereof.

The processing of the plurality of biological samples may include cell-free protein expression. Cell-free protein expression can be used to express toxic proteins. Cell-free protein expression can be used to incorporate unnatural amino acids. Cell-free protein expression may utilize phosphoenolpyruvate, acetyl phosphate, phosphocreatine, or any combination thereof as an energy source. Cell-free protein expression may be performed at ambient temperature, a temperature below ambient temperature (e.g., 0 ℃), a temperature above ambient temperature (e.g., 60 ℃) or any combination thereof.

The processing of multiple biological samples may include preparation for plasmid DNA extraction. Preparation for plasmid DNA extraction may include precipitation of DNA from the lysed cell solution. Preparation for plasmid DNA extraction may include the use of spin column based separation techniques. Preparation for plasmid DNA extraction may include phenol-chloroform extraction.

The processing of the plurality of biological samples may include extracting ribosomes, mitochondria, endoplasmic reticulum, golgi apparatus, lysosomes, peroxisomes, centrosomes, or any combination thereof. Ribosomes, mitochondria, endoplasmic reticulum, golgi apparatus, lysosomes, peroxisomes, centrosomes, or any combination thereof may remain intact.

The processing of the plurality of biological samples may include extracting nucleic acids from the cells. Extracting nucleic acid from a cell may also include extracting long nucleic acid strands, wherein the long nucleic acid strands remain completely intact. The long nucleic acid strand may also be at least 10, 100, 1,000, 10,000, 100,000, 1,000,000 or more base pairs in length. Extraction of nucleic acids may include lysing cells by adding surfactants and detergents such as octyl glucoside, sodium dodecyl sulfate, or octyl phenol polyoxyethylene ether. Extraction of nucleic acids may involve centrifugation, including ultracentrifugation.

The processing of the plurality of biological samples may include sample preparation for mass spectrometry. Sample preparation for mass spectrometry may include cell lysis, digestion, protein amplification, DNA amplification, or other standard sample preparation. Sample preparation for mass spectrometry may include application of the sample to an electrospray ionization (ESI) matrix, integration into a Matrix Assisted Laser Desorption Ionization (MALDI) matrix, or other preparation of ionization. Mass spectrometry can include ion traps, quadrupoles, and other detection methods. The inlet of the mass spectrometer may be directly connected to at least one droplet. The inlet of the mass spectrometer may be adjacent to one or more droplets. Samples for mass spectrometry can be transferred to the inlet of a mass spectrometer by a pipette.

The processing of multiple biological samples may include sample extraction and library preparation for nucleic acid sequencing. Nucleic acid sequencing may include sequencing by synthesis, pyrosequencing, sequencing by hybridization, sequencing by ligation, sequencing by detection of ions released during DNA polymerization, single molecule sequencing, or any combination thereof. Single molecule sequencing may be nanopore sequencing. Single molecule sequencing may be Single Molecule Real Time (SMRT) sequencing.

The processing of the plurality of biological samples may include DNA synthesis using oligonucleotide synthesis, enzyme-catalyzed synthesis, or any combination thereof. Oligonucleotide synthesis may be solid, liquid phase, performed in solution, or any combination thereof. Oligonucleotide synthesis may result in oligonucleotides of at least 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or more nucleotides. The enzyme-catalyzed synthesis may use a polymerase, transferase, other enzymes, or any combination thereof.

The processing of multiple biological samples may include DNA data storage, random access to store DNA, and retrieval of DNA data by DNA sequencing. DNA data storage may utilize DNA strands having greater than about 10, 50, 100, 150, 200, 250, 500, 1,000, 5,000, 10,000, 100,000, and 1,000,000 or more base pairs. DNA sequencing may include at least one PCR reaction, a Maxam-Gilbert sequencer, a Sanger sequencer, or any combination thereof. Nucleic acid sequencing may include sequencing by synthesis, pyrosequencing, sequencing by hybridization, sequencing by ligation, sequencing by detection of ions released during DNA polymerization, single molecule sequencing, or any combination thereof. Single molecule sequencing may be nanopore sequencing. Single molecule sequencing may be Single Molecule Real Time (SMRT) sequencing.

The processing of multiple biological samples may include nucleic acid extraction and sample preparation directly integrated into a sequencer. Nucleic acid extraction and sample preparation may be performed directly on the array. Nucleic acid extraction and sample preparation may be performed adjacent to the array. The sequencer may be adjacent to the array. The sequencer may be coupled to an array. The sequencer may be provided directly on the array.

The processing of the plurality of biological samples may include CRISPR genome editing. Editing may include Cas9 protein, cpf1 endonuclease, crRNA, tracrRNA, or any combination thereof. Repair DNA templates may be used during the editing process. The repair DNA template may be a single stranded oligonucleotide, a double stranded oligonucleotide or a double stranded DNA plasmid.

The processing of the plurality of biological samples may include transcription activator-like effector nuclease (TALEN) genome editing. The processing of the plurality of biological samples may include zinc finger nuclease gene editing.

The processing of the plurality of biological samples may include at least one high throughput process. The high throughput process may be automated so that no input is required. The high throughput process may include at least one assay or characterization method suitable for at least one sample type described herein.

Treatment of multiple biological samples may include screening multiple compounds for multiple cells. The compound may be one or more compounds. The compound may exhibit a biological effect. The biological effect may be promotion or inhibition of cell growth, a signal of the beginning or end of a cellular process, induction of cell division, etc.

The compound may be antibacterial. The antimicrobial chemistry can inhibit bacterial growth by at least 5% to 99% or more. Antibacterial chemicals may kill bacteria.

The compounds may be screened for biological activity. The compounds may use an array of sensors to determine biological activity. For example, a fluorescence detector array can be used to determine the relative amount of fluorescent protein in a biological sample exposed to a compound of interest. Similarly, for example, a microscope may be used to analyze the total number of cell types after exposure to a compound. The compounds may be isolated. The separation process may involve centrifugation, transfer by pipetting or other liquid transfer techniques, precipitation, chromatographic techniques (e.g., column chromatography, thin layer chromatography, etc.), distillation, lyophilization, or recrystallization. Screening for biological activity may include mixing at least one biological sample in at least one droplet with at least one chemical.

The cells may be bacterial cells. Bacterial cells may be pathogenic. Bacterial cells may be resistant to antibiotics. The bacterial cells may be genetically engineered.

The cell may be a eukaryotic cell. Eukaryotic cells may be unicellular organisms (e.g., protozoa, algae), diatoms, fungal cells, insect cells, animal cells, mammalian cells, or human cells. Eukaryotic cells may be derived from unicellular organisms (e.g., protozoa, algae), diatoms, fungi, insects, animals, mammals, or humans. Eukaryotic cells may be derived from larger tissues or organs. Eukaryotic cells may be genetically engineered. Eukaryotic cells may be suspected of having or carrying a disease.

The cells may be prokaryotic cells. Prokaryotic cells may be genetically engineered.

The processing of the plurality of biological samples may include culturing the cells, thereby producing cultured cells. The culturing of the cells may be performed in separate droplets. The culturing of the cells may be performed in separate physical compartments. The culture of the cells can be performed autonomously (without input). The culturing of the cells may be performed on a solid, liquid or semi-solid medium. The culturing of the cells may be performed in two or three dimensions. The culturing of the cells may be performed under ambient or non-ambient conditions (e.g., elevated temperature, low pressure, etc.). The separate physical compartment may be a separate electrowetting chip.

Interactions between the cultured cells or between the cultured cells and at least one biological sample may be determined. Interactions between two or more cultured cell samples can be determined by mixing. The interaction of at least one biological sample with the cultured cells may be determined by mixing, applying the cultured cells directly to the biological sample, or applying the biological sample directly to the cultured cells. Applying the cultured cells may include transferring a liquid cell culture or placing a solid cell culture on a relevant sample.

The cultured cells can be tested on the array or arrays described herein.

The cultured cells may be isolated from the culture. Separation may involve centrifugation, transfer by pipetting or other liquid transfer techniques, precipitation, scraping cells from culture, or chromatographic techniques (e.g., cell chromatography). The isolated cells may be transferred to an external container. The external container may be a biological molecular sieve (SBS) format plate, petri dish, bottle, box, other culture medium, or the like.

Isolated cells can be prepared for nucleic acid sequencing.

Isolated cells can be prepared for protein analysis. The protein assay may be an amino acid assay, a size assay, an absorption assay, a Kjeldahl method, a Dumas method, a Western blot assay, a High Performance Liquid Chromatography (HPLC) assay, a liquid chromatography-mass spectrometry (LC/MS) assay, or an enzyme-linked immunosorbent assay (ELISA) assay.

Isolated cells can be prepared for metabonomic analysis. The metabonomic analysis may be aqueous metabolite analysis, lipid metabolite analysis, nuclear magnetic resonance spectroscopy (NMR) analysis, or mass spectrometry analysis.

The array may include a plurality of lyophilized reagents, dried reagents, stored beads, or any combination thereof. A plurality of lyophilized reagents, dried reagents, stored beads, or any combination thereof may be reconstituted. The lyophilization reagents can include proteins, bacteria, microorganisms, vaccines, drugs, molecular barcodes, oligonucleotides, primers, DNA sequences for hybridization, enzymes (e.g., glucosidase, alcohol dehydrogenase, DNA polymerase, etc.), and dehydration chemicals. The dry reagents may include chemical powders (e.g., salts, metal oxides, etc.), biologically derived chemicals, dry buffer chemicals, other bioactive chemicals, and the like. The stored beads may be magnetic beads, beads for storing bacteria, enzymes, oligonucleotides or molecular sieves. The molecular barcode may be a DNA fragment having at least 5, 10, 20, 30, 40, 50, 60 or more base pairs. The oligonucleotide may be at least 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or more nucleotides. The primer may be DNA or RNA. The DNA sequences used for hybridization can be used to detect minor differences in nucleotide sequence. The DNA sequence may be used in combination with a mismatch detection protein.

The droplet, droplets, derivatives thereof, or any combination thereof may be used to reconstitute a lyophilized reagent, a dried reagent, a stored bead, or any combination thereof. The gel, which may dissolve, suspend or form a lyophilized reagent, a dried reagent, stored beads, or any combination thereof, is reconstituted. The reagents may be preformed as a component of the array.

In some embodiments, the plurality of droplets includes a third droplet comprising a third reagent.

The array may store the plurality of reagents as a solid, liquid, gas, or any combination thereof. The array may condense, sublimate, defrost, evaporate, or any combination thereof, the stored reagents. The reagent may be a compressed gas (e.g., air, argon, nitrogen, oxygen, carbon dioxide, etc.), a solvent (e.g., water, dimethylsulfoxide, acetone, ethanol, etc.), a detergent (e.g., ethanol, SDS, liquid soap, etc.), or a solution (e.g., buffer, chemicals dissolved in a liquid, etc.). In one example of an array physically switching to a storage reagent, solid carbon dioxide (dry ice) may be sublimated to provide cold carbon dioxide gas to the droplets. Another example may be an array boiling water to introduce steam into the droplets or cleaning the array.

The array may dispense a plurality of liquids. The array may dispense a variety of liquids using a variety of methods, such as by pipetting, condensing, decanting, or any combination thereof, using devices such as microfluidic devices, diaphragm pumps, nozzles, piezoelectric pumps, needles, test tubes, acoustic dispensers, capillaries, or any combination thereof. The plurality of liquids may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more liquids.

The array may mix multiple liquids. Mixing may be performed by stirring, sonication, vibration, air flow, bubbling, shaking, vortexing, and electrowetting forces. The plurality of liquids may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more liquids. The liquids may be in the form of at least one droplet. The at least one droplet may be located on an electrowetting array.

The processing of multiple biological samples may be automated (e.g., capable of running without user input). Automation may be run using a program. The program may be a machine learning algorithm. The program may utilize a neural network. Automation may be controlled by the device. The device may be a computer, tablet, smart phone, or any other device capable of executing code. Automation may be interfaced with one or more components of the array (e.g., sensors, liquid handling devices, etc.) to perform a process. In some embodiments, automation may use a camera to track the size of droplets on an array. When the droplet loses sufficient volume due to evaporation, as determined by a computer vision program, the automation will instruct the liquid handling unit to dispense a precise amount of liquid to the droplet to maintain the preset volume. In this embodiment, the open configuration may allow for easier viewing of the droplets. Automated procedures may also perform self-diagnostics using machine learning classifiers to monitor trials for atypical events that may indicate errors. Machine learning algorithms can also be used to improve the performance of automated experiments. Machine learning data may be compiled and analyzed to suggest modifications to control algorithms that may improve trial development.

The array may be reused. The array may have alternative surfaces. The array may have a replaceable membrane. The array may have a replaceable cartridge. The replaceable cartridge may include a membrane. The film may be attached to the array. Vacuum may be used to secure the membrane to the array. An adhesive may be used to couple the membrane to the array. The binder may be non-reactive, pressure sensitive, contact reactive, thermally reactive (e.g., anaerobic, multicomponent (e.g., polyester, polyol, acrylic, etc.), premixed, frozen, one-component), natural, synthetic, or any combination thereof. The adhesive may be applied by spraying, brushing, rolling, or by a film or applicator. The adhesive may be, but is not limited to, silicone, acrylic, epoxy, polyurethane, starch, cyanoacrylate, polyimide, or any combination thereof. The array may be reused at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1,000 times or more. The replaceable surface can be easily removed and reattached to the array. An alternative surface may be a layer of liquid. The liquid may be oil. Alternative films may be polymers (e.g., polyethylene, polytetrafluoroethylene, polydimethylsiloxane, etc.). Alternative films may vary in thickness from 1 nanometer to 1 millimeter. The replaceable cartridge may include a new electrowetting chip. The replaceable cartridge may include a new surface placed over the electrodes of the electrowetting chip.

The array may be washed. The array may be washed in its entirety. The array may be partially purged. The array may be washed using materials stored in the reagent dispensing array. The array may be cleaned using a solid cleaner (e.g., powdered soap, solid antimicrobial, etc.), a liquid cleaner (e.g., liquid soap, ethanol, etc.), or a gaseous cleaner (e.g., steam). About 1% to 100% of the array may be washable.

The array may be disposable. The disposable array may include the entire sample assembly. The disposable array may include a surface of an electrowetting chip. The disposable array can be easily removed.

The volumes of the biomolecules of the array can be manipulated as a mixture. The volume of the biomolecule may include a plurality of nucleic acids, protein sequences, or a combination thereof. Multiple nucleic acids, protein sequences, or combinations thereof can be manipulated by modulating local surface charges without physical contact with the mixture through another component of the array. For example, an electrowetting chip can be used to move droplets containing a large amount of nucleic acid by changing the surface wetting properties of the droplets. Thus, the droplet may move without contacting another component of the array. The mixture may be within a droplet. The droplets can include a volume of at least 1 picoliter (pL), 10pL, 100pL, 1 nanoliter (nL), 10nL, 100nL, 1 μl, 10 μl, 100 μl, 1 milliliter (mL), 10mL, or more. The mixture may comprise a protein having DNA ligase activity. The mixture may comprise a protein having DNA transposase activity. Proteins having DNA ligase activity may be derived from viruses (e.g., T4), bacteria (e.g., e.coli), or mammals (e.g., human DNA ligase 1). Proteins having DNA transposase activity may be derived from bacteria (e.g., tn 5) or mammals (e.g., sleeping Beauty (SB) transposase). The volume of the biomolecules tested can be manipulated by a lateral geospatial movement of the mixture of at least 1 mm. The volume of the biomolecule being tested can be manipulated by a specified or pre-specified set of instructions. The instructions may be associated with a particular location of the array.

The array may comprise reagents for performing a strand displacement amplification reaction, a self-sustained sequence replication and amplification reaction, or a Q3 replicase amplification reaction. The reagents for performing the strand displacement amplification reaction may be Bst DNA polymerase, cas9 or another form of nicking protein in the form of a semi-phosphorothioate. The self-sustaining sequence replication and amplification reagents may be Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (RT), escherichia coli RNase H, T7 RNA polymerase, or any combination thereof. The reagents for the Q3 replicase amplification reaction may be derived from Q3 phage, e.coli, or any combination thereof.

The array may include reagents including DNA ligases, nucleases, or restriction endonucleases. The DNA ligase may be derived from a virus (e.g., T4), a bacterium (e.g., e.coli), or a mammal (e.g., human DNA ligase 1). The nuclease may be an exonuclease (digestion starting from the molecular end) or an endonuclease (digestion starting from a place other than the molecular end). The nuclease may be a deoxyribonuclease (acting on DNA) or a ribonuclease (acting on RNA). The restriction endonuclease may be a I, II, III, IV or V-type restriction endonuclease. One example of a restriction endonuclease may be cas9 or a zinc finger nuclease.

The array may include reagents for preparing amplified nucleic acid products. The reagents for preparing the amplified nucleic acid product may be Bst DNA polymerase, deoxyribonucleotide triphosphates, fragments of e.coli DNA polymerase 1, avian myeloblastosis virus reverse transcriptase, RNase H, T7 DNA dependent RNA polymerase, taq polymerase, other DNA polymerase/transcriptases, or any combination thereof.

The array may be a component in the manufacture of a kit or system for the diagnosis or prognosis of a disease. The kit may process a biological sample. The biological sample may be a sample from a patient. In some embodiments, the array may be used to process samples from patients suspected of having a disease. The disease may be a disease classified by the center for disease control and prevention (CDC). The array may mix the sample with the reagent. The array may mix the sample with reagents for separating cells from serum. The array may process cells or derivatives thereof. The array may transfer the cells or derivatives thereof to an optical device coupled to the array. The cells or derivatives thereof may be treated according to the methods described herein.

The array may comprise a protein having nucleic acid cleavage activity. The array may comprise biomolecules having RNA cleavage activity. The protein having nucleic acid cleavage activity may be a ribonuclease, a deoxyribonuclease, or any combination thereof. The biological molecule having RNA cleavage activity may be a small ribonuclease, a large ribonuclease, or any combination thereof.

A set of interchangeable reagents can be introduced via at least one solid support. The solid support may be a paper tape. The solid support may be a microbead. The solid support may be a column. The support post may be attached to the base of the carrier or may be integral with the carrier. The solid support may be a microwell. The solid support may be a slide, a spoon or a plastic film. The solid support may be a bead. The beads may be magnetic. The interchangeable reagent sets may be chemical reagents (e.g., small molecules, metals, etc.), biological species (e.g., proteins, DNA, RNA, etc.), processing reagents (e.g., PCR reagents, etc.).

The interchangeable reagent set may be introduced via at least one second carrier. The second support may be a microwell. The second carrier may be an SBS plate, petri dish, bottle, slide or other container. The interchangeable reagent sets may be chemical reagents (e.g., small molecules, metals, etc.), biological species (e.g., proteins, DNA, RNA, etc.), processing reagents (e.g., PCR reagents, etc.).

The array may contain a template independent polymerase. The template independent polymerase may be a terminal deoxynucleotidyl transferase (TdT). The array may include an enzyme that restricts nucleic acid polymerization. The enzyme that limits nucleic acid polymerization may be apyrase. The array may have a sensor to detect the presence of at least one terminal "C" tail in the nucleic acid molecule. The at least one terminal "C" tail may be isolated. Apyrase may be derived from E.coli, potato or arthropod.

Multiple biological samples of the array may be stored by drying. Drying may be performed by heating, vacuum, flowing gas, lyophilization, or any combination thereof. The sample may be stored on an array or in other containers. Other containers may be slides, petri dishes, culture medium bottles, test tubes or (micro) well arrays.

Multiple biological samples of the array may be recovered by means of rehydration. Rehydration may be performed by adding a liquid to the dried multiple biological samples or by blowing a gas containing a liquid into the dried multiple biological samples. The rehydrated plurality of biological samples may be manipulated using any of the liquid handling mechanisms described above.

The plurality of biological samples may be deposited in SBS format on the plurality of arrays, or on any random location of the plurality of arrays, thereby producing at least one deposited biological sample. The SBS format may be a 96-well plate size. The deposited biological sample may be a solid or a liquid.

In preparation forIn manipulating samples on a chip, a commercial acoustic liquid handler may be used to deposit multiple biological samples. The acoustic liquid processor may beOr ATS->The at least one deposited biological sample may be used for cell-free synthesis. The at least one deposited biological sample may be used in combination to assemble large DNA constructs. The assembled large DNA construct may be assembled by Gibson assembly, circular polymerase extension cloning and DNA assembly.

The processing of the plurality of biological samples may include at least one of the following assays or any combination thereof: digital PCR, isothermal amplification of nucleic acids, antibody-mediated detection, enzyme-linked immunosorbent assay (ELISA), electrochemical detection, colorimetric assay, fluorescent assay, and micronuclear assay.

Digital PCR assays can handle droplets of up to about 1,000 microliters, 900 microliters, 800 microliters, 700 microliters, 600 microliters, 500 microliters, 400 microliters, 300 microliters, 200 microliters, 100 microliters, 50 microliters, 10 microliters, 1 microliter, 0.1 microliters, 0.01 microliters, 0.001 microliters, 0.0001 microliters, or less. Digital PCR can use at least about 0.0001 microliter, 0.001 microliter, 0.01 microliter, 0.1 microliter, 1 microliter, 10 microliters, 50 microliter, 100 microliter, 200 microliter, 300 microliter, 400 microliter, 500 microliter, 600 microliter, 700 microliter, 800 microliter, 900 microliter, 1,000 microliter, or more of the initial droplets. Digital PCR can use about 100 microliters to about 1 microliter of initial droplet. Digital PCR can use about 50 microliters to about 1 microliter of initial droplet. In some embodiments, the digital PCR assay can separate a droplet or droplets to form at least about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or more droplets. The droplet or droplets may be separated by oil-water emulsification techniques.

Isothermal amplification of nucleic acids may be PCR, strand Displacement Amplification (SDA), rolling Circle Amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic Acid Sequence Based Amplification (NASBA), helicase Dependent Amplification (HDA), recombinase Polymerase Amplification (RPA), cross Primer Amplification (CPA), or any combination thereof.

Antibody-mediated detection may be used to detect cells, proteins, nucleic acid molecules (e.g., DNA, RNA, PNA, etc.), hormones, antibodies, small molecules, or any combination thereof. The antibody-mediated detection may include antibodies comprising antigen binding sites that specifically detect cells, proteins, nucleic acids, or any combination thereof. The antibody may be of natural origin. The antibody may be a synthetic antibody. The synthetic antibody may be a recombinant antibody, a nucleic acid aptamer, a non-immunoglobulin scaffold, or any combination thereof.

The enzyme-linked immunosorbent assay (ELISA) may be of the direct type, sandwich type, competitive type, reverse type or any combination thereof. ELISA can detect, quantify, or a combination of detect, quantify various substances, such as peptides, proteins, antibodies, hormones, small molecules, or any combination thereof.

The electrochemical detection may be an oxidation or reduction based electrochemical detection. Electrochemical detection based on oxidation or reduction may be conductivity, potentiometry, voltammetry, amperometry, coulometry, impedance or any combination thereof. Electrochemical detection may be used to detect cells, proteins, nucleic acids, hormones, small molecules, antibodies, or any combination thereof. Electrochemical detection can detect the current generated by the oxidation or reduction reaction of a biological sample. Electrochemical detection can detect the current generated by the oxidation or reduction reaction of a biological sample.

Colorimetric assays may be used to detect cells, nucleic acids, proteins, small molecules, antibodies, hormones, or any combination thereof. Colorimetric assays may be used to determine absorbance at least at wavelengths of 240nm, 280nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, 1250nm, 1500nm, 1750nm, 2000nm, 2400nm or greater. Colorimetric assays may be used to determine absorbance at wavelengths of up to 2400nm, 2000nm, 1750nm, 1500nm, 1250nm, 1000nm, 950nm, 900nm, 850nm, 800nm, 750nm, 700nm, 650nm, 600nm, 550nm, 500nm, 450nm, 400nm, 350nm, 300nm, 280nm, 240nm or less. Colorimetric assays may be used to determine absorbance at wavelengths of about 2400nm to about 240 nm. Colorimetric assays may be used to determine absorbance at wavelengths of about 1000nm to about 100 nm. Colorimetric assays may be used to determine absorbance at wavelengths of about 900nm to about 400 nm. Colorimetric tests may be performed on solid, liquid or gas samples. Colorimetric tests may use broadband light sources (e.g., incandescent light sources, LEDs, etc.), laser light sources, or combinations thereof. The light source may pass through various optical elements (e.g., lenses, filters, mirrors, etc.) before and after interacting with the sample. The transmitted or reflected light may be detected by a Charge Coupled Device (CCD), photomultiplier tube, avalanche photodiode, or any combination thereof (e.g., by a mirror, optical fiber, etc.). The detector may be coupled to a wavelength selective device, for example a monochromator or a filter bank.

Fluorescence assays can be used to detect cells, nucleic acids, proteins, small molecules, antibodies, hormones, or any combination thereof. Fluorescence assays can be used to determine absorbance at least at wavelengths of 240nm, 280nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, 1250nm, 1500nm, 1750nm, 2000nm, 2400nm or greater. Fluorescence assays can be used to determine absorbance at wavelengths of up to 2400nm, 2000nm, 1750nm, 1500nm, 1250nm, 1000nm, 950nm, 900nm, 850nm, 800nm, 750nm, 700nm, 650nm, 600nm, 550nm, 500nm, 450nm, 400nm, 350nm, 300nm, 280nm, 240nm or less. Fluorescence assays can be used to determine emissions at wavelengths of about 2400nm to about 240 nm. Fluorescence assays can be used to determine emissions at wavelengths of about 1000nm to about 100 nm. Fluorescence assays can be used to determine emissions at wavelengths from about 900nm to about 400 nm. The fluorescence test may use a broadband light source (e.g., incandescent light source, LED, etc.), a laser light source, or a combination thereof. The light source may pass through various optical elements (e.g., lenses, filters, mirrors, etc.) before and after interacting with the sample. Fluorescence may be detected by a CCD, photomultiplier tube, avalanche photodiode, or any combination thereof. The detector may be coupled to a wavelength selective device such as a monochromator or a filter or filter bank. For example, a fluorescent assay can be used to determine the concentration of reduced NADPH, as NADPH fluoresces in its reduced form rather than in its oxidized form. In this example, the observed change in fluorescence intensity over time corresponds linearly to the amount of reduced NADPH in the sample.

Micronucleus assays can assess the presence of micronuclei in biological samples. Micronuclei may contain chromosomal fragments resulting from DNA fragmentation (chromosome-scission precursors) or intact chromosomes resulting from disruption of the mitotic machinery (aneuploidy). Micronuclear assays can be used to identify genotoxic compounds. Genotoxic compounds may be carcinogens. Micronucleus assays may be performed in vivo or in vitro. In vivo micronucleus assays may utilize bone marrow or peripheral blood from biological samples. In vitro micronucleus assays may utilize cells or tissues derived from multiple biological samples.

The processing of the plurality of biological samples may include isothermal amplification of at least one selected nucleic acid or polynucleotide, which may include: providing at least one sample by combining droplets containing a plurality of reagents that are effective to allow at least one isothermal amplification reaction of the sample without mechanical manipulation, the sample may comprise at least one nucleic acid; and performing at least one isothermal amplification reaction to amplify the nucleic acid.

The at least one isothermal amplification of the at least one selected nucleic acid may be PCR, strand Displacement Amplification (SDA), rolling Circle Amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic Acid Sequence Based Amplification (NASBA), helicase Dependent Amplification (HDA), recombinase Polymerase Amplification (RPA), cross Primer Amplification (CPA), or any combination thereof. The at least one isothermal amplification may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more isothermal amplifications.

The combined droplets may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more droplets. The plurality of reagents may be any of the isothermal amplification reagents described herein.

The processing of the plurality of biological samples may include means for detecting a Polymerase Chain Reaction (PCR) product on at least one droplet. The droplets may be aqueous droplets. The device can: generating at least one droplet comprising a plurality of nucleic acid and protein molecules on an electrowetting array; performing a PCR reaction in the presence of aqueous droplets on the array surface; and interrogating the droplet with a detector. The PCR product may be DNA or RNA. The protein molecule may be an enzyme, used in a PCR reaction, or used to report the progress of a reaction (e.g., luminescence). The performance of the PCR reaction may include agitating the sample (e.g., stirring, vibration, electrowetting-based movement, etc.), heating or cooling the sample (using the heater and cooler arrays described above), and controlling droplet size. The detector may be any of the detectors described herein.

The device may comprise a plurality of reporter molecules. The reporter may be a fluorescent reporter. During the PCR reaction, the plurality of fluorescent reporter molecules may be separated from the at least one quencher molecule by at least one enzyme. The at least one enzyme may comprise a polymerase, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase. The plurality of fluorescent reporter molecules may be proteins, luminescent small molecules, luminescent nucleic acids, or nanoparticles.

The nucleic acid may be detected by a sensor. The sensor may detect the radiolabel. The sensor may detect the fluorescent marker. The sensor may detect a chromophore. The sensor may detect the redox marker. The sensor may be a p-n type diffusion diode. Nucleic acids may be detected by a smart phone.

The processing of the plurality of biological samples may include binding at least one biological molecule on the array. At least one biomolecule may be immobilized on the surface. At least one biomolecule may be immobilized on a diffusible matrix. At least one biomolecule may be immobilized on a diffusible bead. The at least one biomolecule may be a protein, a compound derived from a biological system (e.g., a signaling molecule, cofactor, etc.), a drug, a molecule that exhibits or is suspected to exhibit biological activity, a carbohydrate, a lipid, a nucleic acid, a natural product, or a nutrient. Immobilization may be by adsorption, ionic interactions, covalent bonding or intercalation. The surface may be an electrowetting chip, a polymer, a dielectric, a metal, a fiber-based sheet (e.g., paper tape), or a stationary phase (e.g., silica gel). The diffusible matrix may be a polymer, a tissue (e.g., a gel), or an aerogel. The diffusible beads may be polymer beads, molecular sieves or beads formed from biological materials (e.g., beaded proteins or nucleic acids). The location of the biomolecule may be identified by a coding scheme. The coding scheme may be a pre-programmed method for determining the location of the biomolecules. The coding scheme may be based on the chemical structure to which it is fixed.

In some embodiments, the detectable label may be a fluorescent label that emits a particular wavelength. In some embodiments, the fluorescent label emits light when excited by a light source. In some embodiments, the detectable label emits light having a wavelength of 380-450 nm. In some embodiments, the detectable label emits light at wavelengths of 450-495 nm. In some embodiments, the detectable label emits light at a wavelength of 495-570 nm. In some embodiments, the detectable label emits light at a wavelength of 570-590 nm. In some embodiments, the detectable label emits light having a wavelength of 590-620 nm. In some embodiments, the detectable label emits light having a wavelength of 620-750 nm. In some embodiments, the computer vision system uses interchangeable filters. In some embodiments, the optical filter is used in conjunction with one or more optical sensors or image sensors of a computer vision system. In some embodiments, a filter is provided to filter the wavelengths generated by the detectable labels such that only one or more labels corresponding to a particular type of sample are detected or monitored by the system. In some embodiments, the system may include one or more optical sensors, where each optical sensor is configured with a particular filter to monitor a particular marker corresponding to a particular type of sample as described herein.

In some embodiments, the array can induce interactions of multiple biomolecules from two or more discrete liquid volumes without mechanical manipulation. Such interactions may be mixing, chemical reactions, adsorption or enzymatic reactions. The absence of mechanical manipulation may mean that the interacting moving parts may be two or more discrete liquid volumes. The plurality of biological molecules may be at least one of a protein, a compound derived from a biological system (e.g., a signaling molecule, cofactor, etc.), a drug, a molecule that exhibits or is suspected to exhibit biological activity, a carbohydrate, a lipid, a nucleic acid, a natural product, or a nutrient.

The array can produce amplified nucleic acid products without mechanical manipulation. The array can perform diagnostic tests on nucleic acid samples without mechanical manipulation. The array can perform diagnostic or prognostic tests on biological samples without mechanical manipulation. Multiple biological samples may be suspected of containing a nucleic acid biomarker.

The array may include a gas source contacting and absorbable by the at least one droplet. At least one droplet may be manipulated on the device. The gas may be air, nitrogen, argon, carbon dioxide, hydrogen or water vapor. At least one droplet may absorb at least 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more of the gas. The manipulation may be due to the pressure exerted by the gas on the at least one droplet.

The plurality of biological samples may include reagents for performing a strand displacement amplification reaction, a self-sustaining sequence replication, an amplification reaction, or a Q3 replicase amplification reaction. The reagents for performing the strand displacement amplification reaction may be Bst DNA polymerase, cas9 or other phosphorohalidate form of nicking protein. The self-sustaining sequence replication and amplification reagents may be Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (RT), escherichia coli RNase H, T7 RNA polymerase, or any combination thereof. The reagents for the Q3 replicase amplification reaction may be derived from Q3 phage, e.coli, or any combination thereof.

The array may receive at least one instruction from a remote computer to process the biological sample array. At least one instruction may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more instructions. The remote computer may be any system capable of sending instructions (e.g., desktop, notebook, tablet, smart phone, application specific integrated circuit, etc.). The remote computer may not require user input to send the at least one instruction.

The array may be pre-programmed to process the biological sample array. The pre-programming may be used for processing of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more steps. The pre-programming may be stored in an array (e.g., in a hard disk drive, flash memory unit, erasable programmable read-only memory (EPROM), tape cartridge, etc.), or in an additional system capable of sending instructions (e.g., desktop, notebook, tablet, smart phone, application specific integrated circuit, etc.).

The array may receive information related to the DNA sequence. Information related to a DNA sequence may include the length of the DNA sequence, the composition of the DNA sequence (e.g., the total number of given bases, the sequence of bases, etc.), or the presence of a particular DNA sequence. The DNA sequence may trigger an automated process. Information related to DNA sequences may trigger automated processes. The automated process may include converting the DNA sequence into at least one constituent oligonucleotide sequence. The at least one constituent oligonucleotide sequence may be assembled, error corrected, recombined or any combination thereof into a DNA amplicon. The DNA amplicon may direct the production of RNA, protein, biological particles, or any combination thereof. The biological particles may be derived from a virus.

The array may produce at least one peptide or antibody from a DNA template. The array may be produced using in vivo methods (e.g., using cell production) or cell-free production (e.g., production without living organisms). The peptide may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids. The amino acids may be naturally occurring or non-naturally occurring. Antibodies may be surface bound or free. Antibodies may be derived from any of a variety of biological samples.

The array may divide the at least one droplet into a plurality of droplets by electrodynamic forces, electrowetting forces, dielectric electrowetting forces, dielectrophoresis effects, acoustic forces, hydrophobic knives, or any combination thereof. Electrowetting forces may be induced by the configuration of the array described above. The dielectrophoresis effect may be light-induced (electromagnetic radiation may be used to induce this effect). The dielectrophoresis effect may be induced by wires, sheets, electrodes, or any combination thereof produced by photolithography, laser ablation, electron beam patterning, or any combination thereof. The wires, sheets, and electrodes may be made of metals (e.g., gold, copper, silver, titanium, etc.), metal alloys, semiconductors (e.g., silicon, gallium nitride), or conductive oxides (e.g., indium tin oxide). The acoustic force may be ultrasonic. The acoustic force may be generated by a sensor. The hydrophobic knife may be a hydrophobic microtome or a hydrophobic blade.

The partitioning process may dispense reagents. The agent may be any of the agents described herein.

The segmentation process may assign samples. The sample may be a plurality of biological samples. The sample may be a non-biological sample (e.g., a chemical).

The reaction can be carried out by mixing separate droplets. The reaction may be an amplification reaction, a chemical conversion, a binding reaction, a reaction of an antimicrobial agent with a microorganism, or a reaction as described above.

The segmented droplets may be analyzed using a sensor. The sensor may be any of the sensors in the sensor arrays described above.

The segmented droplets may be mixed with at least one target droplet to keep the volume of the at least one target droplet constant. The constant volume may be determined by computer vision (coupled camera and algorithm), mass, or optical spectroscopy (e.g., absorption spectroscopy).

The array may process multiphase fluids. The fluid may have at least 2, 3, 4, 5, 6 or more phases. For example, a drop of water containing a colloid will have 3 phases when itself surrounded by a drop of oil.

The array may use dielectrophoretic force (DEP) for cell sorting, cell separation, manipulation of at least one bead, or any combination thereof. DEP may be light-induced (electromagnetic radiation may be used to induce this effect). The DEP may be induced by wires, sheets, electrodes, or any combination thereof produced by photolithography, laser ablation, electron beam patterning, or any combination thereof. The wires, sheets, and electrodes may be made of metals (e.g., gold, copper, silver, titanium, etc.), metal alloys, semiconductors (e.g., silicon, gallium nitride), or conductive oxides (e.g., indium tin oxide). The beads may include magnetic beads, beads for storing bacteria, enzymes, oligonucleotides, nucleic acids, antibodies, PCR primers, ligands, molecular sieves, or any combination thereof. Sorting and isolation may be used to pre-concentrate at least one cell in the original clinical sample. The original clinical sample may be derived from a plurality of biological samples. The original clinical sample may be from a subject suffering from or suspected of suffering from a disease.

The biological sample or samples may be deposited on an array or arrays. The plurality of arrays may include at least two arrays. One of the plurality of arrays may include a surface. The surface may comprise glass, polymer, ceramic, metal, or any combination thereof. The surface may comprise an EWOD array, DEW array, DEP array, microfluidic array, or any combination thereof. The plurality of arrays may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more arrays. The plurality of arrays may comprise up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arrays. The plurality of arrays may include 1,000 to 2 arrays, 500 to 100 arrays, 100 to 2 arrays, 100 to 50 arrays, 50 to 2 arrays, 50 to 10 arrays, or 10 to 2 arrays. One of the plurality of arrays may be adjacent to another of the plurality of arrays. These arrays may be horizontally, vertically or diagonally adjacent.

The surface may have a thickness of at most 1,000 μm, 500 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm or less. The surface may have a thickness of at least 0.01 μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 500 μm, 1,000 μm or more. The thickness of the surface may be 1,000 μm to 0.01 μm, 500 μm to 1 μm, 100 μm to 1 μm or 50 μm to 1 μm.

The surface may have a roughness of maximum 1,000 μm, 500 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm, 0.001 μm or less. The surface may have a roughness of at least 0.001 μm, 0.01 μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 500 μm, 1,000 μm or more. The roughness of the surface may be 1,000 μm to 0.001 μm, 500 μm to 0.01 μm, 100 μm to 1.0 μm or 50 μm to 1.0 μm.

The surface may comprise a liquid layer having wet affinity properties for the surface. The liquid may be immiscible with the one or more droplets. The liquid may be dispensed on a surface. The upper surface of the liquid may reduce friction between the liquid droplet or droplets and the surface compared to a liquid droplet direct contact surface.

The plurality of arrays may comprise channels, wells, or any combination thereof. The plurality of arrays may comprise a plurality of channels, a plurality of wells, or any combination thereof. The channel or channels may pass between at least one surface. A gas, liquid, solid, or any combination thereof may be transported through the channel or aperture. The gas, liquid, solid, or any combination thereof may be transported through a plurality of channels or a plurality of holes. Gas, liquid, solid, or any combination thereof may be transferred from one array to another. These arrays may be adjacent to each other. A gas, liquid, solid, or any combination thereof may be transferred from one array to at least one other array. A gas, liquid, solid, or any combination thereof may be transferred from one array to at least two, three, four, five, six, seven, eight, nine, ten, or more arrays.

At least two droplets of the plurality of droplets may be separated by at least one membrane. The membrane may include a metal, a ceramic (e.g., alumina, silicon carbide, zirconia, etc.), a homogeneous membrane (e.g., a polymer (e.g., cellulose acetate, nitrocellulose, cellulose ester, polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, etc.), a heterogeneous solid (e.g., a polymer mixture, mixed glass, etc.), a liquid (e.g., an emulsion liquid membrane, a fixed (supporting) liquid membrane, a molten salt, a liquid membrane containing hollow fibers, etc.), or any combination thereof. The membrane may allow molecules, ions, or a combination thereof to penetrate from one side of the membrane to the other. The membrane may be impermeable, semi-permeable, or a combination thereof. The permeability may be distinguished by size, solubility, charge, affinity, or a combination thereof. The membrane may be porous or semi-porous. The membrane may be a biological membrane, a synthetic membrane, or a combination thereof. The membrane may facilitate exchange of components in one droplet with components in another droplet. The membrane may facilitate passive diffusion, active diffusion, passive transport, active transport, or any combination thereof. The membrane may be a cation exchange membrane, a charge mosaic membrane, a bipolar membrane, an anion exchange membrane, a basic anion exchange membrane, a proton exchange membrane, or a combination thereof. The membrane may be permanently or temporarily attached to the array or arrays.

Reaction time

Some aspects of the present disclosure provide a method of producing a biopolymer, wherein the reaction time is 30 minutes or less. In some embodiments, the reaction time is from about 1 minute to about 30 minutes. In some embodiments of the present invention, in some embodiments, the reaction time is from about 1 minute to about 2 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 15 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 25 minutes, from about 1 minute to about 30 minutes, from about 2 minutes to about 3 minutes, from about 2 minutes to about 4 minutes, from about 2 minutes to about 5 minutes, from about 2 minutes to about 10 minutes, from about 2 minutes to about 15 minutes, from about 2 minutes to about 20 minutes, from about 2 minutes to about 25 minutes, from about 2 minutes to about 30 minutes, from about 3 minutes to about 4 minutes, from about 3 minutes to about 5 minutes, from about 3 minutes to about 10 minutes, from about 3 minutes to about 15 minutes, from about 3 minutes to about 20 minutes, from about 3 minutes to about 25 minutes about 3 minutes to about 30 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 10 minutes, about 4 minutes to about 15 minutes, about 4 minutes to about 20 minutes, about 4 minutes to about 25 minutes, about 4 minutes to about 30 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 25 minutes, about 5 minutes to about 30 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, or about 25 minutes to about 30 minutes. In some embodiments, the reaction time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, the reaction time is at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 25 minutes. In some embodiments, the reaction time is up to about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, the reaction time is about 10 minutes.

Aspects of the present disclosure provide for the generation of at least one nucleic acid molecule of a polynucleotide within a combined droplet in 30 minutes or less. In some embodiments, the reaction time within the combined droplets is from about 1 minute to about 30 minutes. In some embodiments, the reaction time within the combined droplets is from about 1 minute to about 2 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 15 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 25 minutes, from about 1 minute to about 30 minutes, from about 2 minutes to about 3 minutes, from about 2 minutes to about 4 minutes, from about 2 minutes to about 5 minutes, from about 2 minutes to about 10 minutes, from about 2 minutes to about 15 minutes, from about 2 minutes to about 20 minutes, from about 2 minutes to about 25 minutes, from about 2 minutes to about 30 minutes, from about 3 minutes to about 4 minutes, from about 3 minutes to about 5 minutes, from about 3 minutes to about 10 minutes, from about 3 minutes to about 15 minutes, from about 3 minutes to about 20 minutes, from about 3 minutes to about 30 minutes, from about 4 minutes to about 5 minutes, from about 4 minutes to about 10 minutes, from about 4 minutes to about 4 minutes, about 15 minutes, from about 4 minutes to about 4 minutes, from about 20 minutes to about 20 minutes, from about 10 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 5 minutes, from about 10 minutes to about 25 minutes, from about 10 minutes, from about 15 minutes to about 15 minutes. In some embodiments, the reaction time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, the reaction time is at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 25 minutes. In some embodiments, the reaction time within the combined droplets is at most about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, the reaction time within the combined droplets is about 10 minutes.

Washing step

Some aspects of the present disclosure provide one or more cleaning steps. In some embodiments, the one or more washing steps include moving wash droplets to contact the combined droplets. In some embodiments, vibration is applied to one or more cleaning steps.

dNTP

Some aspects of the present disclosure provide droplets or reagents comprising deoxynucleoside triphosphates (dntps). In some embodiments, the first droplet or reagent comprises deoxynucleoside triphosphates (dntps). In some embodiments, the second droplet or reagent comprises deoxynucleoside triphosphates (dntps). In some embodiments, the third droplet or reagent comprises deoxynucleoside triphosphates (dntps). In some embodiments, the combined droplets or reagents comprise deoxynucleoside triphosphates (dntps). In some embodiments, a deoxynucleoside triphosphate (dNTP) may have a protecting group. In some embodiments, the protecting group may be removed in the reaction.

User experience

In some aspects of the disclosure, the user experience may include certain workflow steps. In some embodiments, the user loads a proprietary Volta consumable cartridge for each batch run. In some embodiments, for each batch run, the user loads the reagents into the dispenser. In some embodiments, the user loads samples for each batch run. In some embodiments, the sample is loaded by a pipette. In some embodiments, the user uses the touch screen interface to select his workflow and any other parameters at the beginning of the run. In some embodiments, the user unloads samples during the batch when the batch is complete, or if offline processing is required. In some embodiments, the sample is unloaded using a pipette.

Subsystem

Some aspects of the present disclosure provide a subsystem. In some embodiments, the subsystem may handle four reactions simultaneously. In some embodiments, the reaction includes electrowetting, magnetism, other mechanical degrees of freedom, or a combination thereof. In some embodiments, the instrument may comprise a subsystem. In some embodiments, the instrument may comprise two subsystems. In some embodiments, the instrument can handle four reactions simultaneously. In some embodiments, the instrument can handle eight reactions simultaneously.

Computer hardware

Some aspects of the present disclosure provide for the use of computer hardware. In some implementations, the hardware includes one or more processors described herein. In some implementations, one or more processors described herein are integrated modules. In some embodiments, one or more processors described herein areJetson Nano ^TM Developer(s)A kit processor.

Computer system

The various processes described herein may be implemented by suitably programmed general purpose computers, special purpose computers, and computing devices. Typically, a processor (e.g., one or more microprocessors, one or more microcontrollers, one or more digital signal processors) will receive instructions (e.g., from a memory or similar device) and execute the instructions, thereby performing one or more processes defined by the instructions. The instructions may be embodied as one or more computer programs, one or more scripts, or other forms. The processes may be performed on one or more microprocessors, central Processing Units (CPUs), computing devices, microcontrollers, digital signal processors, or the like, or any combination thereof. The data of the programs and operations to perform the processing may be stored and transmitted using various media. In some cases, hardwired circuitry or custom hardware may be used in place of or in combination with some or all of the software instructions that may implement these processes. Algorithms other than the described algorithm may also be used.

The program and data may be stored in various media suitable for the purpose or may be stored in a combination of heterogeneous media readable and/or written by a computer, processor, or similar device. The media may include non-volatile media, optical or magnetic media, dynamic Random Access Memory (DRAM), static RAM, floppy disk, hard disk, magnetic tape, any other magnetic media, CD-ROM, DVD, any other optical media, punch cards, paper tape, any other physical media with patterns of holes, RAM, PROM, EPROM, FLASH-EEPROM, any other memory chip or cartridge, or other memory technology. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a system bus coupled to the processor.

The database may be implemented using a database management system or a dedicated memory organization scheme. Other database structures than those described may be readily employed. The databases may be stored locally or remotely from a device accessing the data of such databases.

In some cases, the process may be performed in a network environment that includes a computer in communication with one or more devices (e.g., via a communication network). The computer may communicate with the device directly or indirectly via any wired or wireless medium (e.g., the internet, a LAN, a WAN, or ethernet, a token ring, a telephone line, an electrical cable, a radio channel, an optical communication line, a commercial online service provider, a bulletin board system, a satellite communication link, or a combination thereof). Each device itself may comprise a computer or other computing device, such as based on Or is fast ^TM Computing devices of the processor, the devices being adapted to communicate with a computer. Any number and type of devices may be in communication with the computer.

A server computer or centralized management mechanism may or may not be necessary or desirable. In various cases, the network may or may not include a centralized management device. The various processing functions may be performed on a centralized management server, one of several distributed servers, or other distributed devices

The present disclosure provides a programmed computer system for implementing the methods of the present disclosure. Fig. 2 illustrates a computer system 1301, the computer system 1301 being programmed or otherwise configured to manipulate a droplet or droplets on the systems described herein. Computer system 1301 can adjust various aspects of sample operation of the present disclosure, e.g., droplet size, droplet volume, droplet position, droplet velocity, droplet wetting, droplet temperature, droplet pH, beads in a droplet, number of cells in a droplet, droplet color, concentration of chemical material, concentration of biological substance, or any combination thereof. The computer system 1101 may be the user's electronic device or may be a computer system that is remotely located from the electronic device. The electronic device may be a mobile electronic device.

Computer system 1301 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 1305, which may be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 1301 also includes memory or memory locations 1310 (e.g., random access memory, read only memory, flash memory), electronic storage 1315 (e.g., hard disk), a communication interface 1320 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1325 such as cache, other memory, data storage, electronic display adapter, or any combination thereof. The memory 1310, storage unit 1315, interface 1320, and peripheral 1325 communicate with the CPU 1305 through a communication bus (solid line) such as a motherboard. Storage unit 1315 may be a data storage unit (or data repository) for storing data. Computer system 1301 can be operably connected to a computer network ("network") 1330 via a communication interface 1320. The network 1330 may be the Internet, an extranet, or any combination thereof, or an intranet in communication with the Internet, an extranet, or any combination thereof. In some cases, the network 1330 is a telecommunications network, a data network, or any combination thereof. The network 1330 may include one or more computer servers that may implement distributed computing, such as cloud computing. In some cases, with the aid of computer system 1301, network 1330 may implement a point-to-point network that may enable devices coupled to computer system 1301 to act as clients or servers.

The CPU 1305 may execute a series of machine readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 1310. These instructions may be directed to the CPU 1305, which CPU 1305 may then program or otherwise configure the CPU 1305 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1305 may include fetch, decode, execute, and write back.

The CPU 1305 may be part of a circuit such as an integrated circuit. One or more other components of system 1101 may be included in the circuit. In some cases, the circuit is an Application Specific Integrated Circuit (ASIC).

The storage unit 1315 may store files such as a driver, a library, and a saved program. The storage unit 1315 may store user data, such as user preferences and user programs. In some cases, computer system 1301 may include one or more additional data storage units external to computer system 1301, such as a storage unit located on a remote server in communication with computer system 1301 via an intranet or the internet.

Computer system 1301 can communicate with one or more remote computer systems over network 1330, e.g., computer system 1301 can communicate with a user's remote computer system (e.g., mobile electronic device). Examples of remote computer systems include personal computers (e.g., portable PCs), tablet computers (e.g., Galaxy Tab), phone, smart phone (e.g.,android-supporting device, blackberry +.>) Or a personal digital assistant. A user may access computer system 1301 via network 1330.

The methods described herein may be implemented by machine (e.g., a computer processor) executable code stored on an electronic storage location of computer system 1301, such as memory 1310 or electronic storage unit 1315. The machine executable code or machine readable code may be provided in the form of software. During use, code may be executed by the processor 1305. In some cases, code may be retrieved from storage 1315 and stored in memory 1310 for ready access by processor 1305. In some cases, the electronic storage unit 1315 may be eliminated and the machine-executable instructions stored in the memory 1310.

The code may be precompiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled at runtime. The code may be provided in a programming language, which may be selected to enable the code to be executed in a precompiled or compiled manner.

Some aspects of the systems and methods provided herein, such as computer system 1301, may be embodied in programming. Aspects of the technology may be considered "an article of manufacture" or "an article of manufacture" which typically exists in the form of machine (or processor) executable code, associated data, or any combination thereof, which is carried or embodied in a type of machine-readable medium. The machine executable code may be stored in an electronic storage unit such as a memory (e.g., read only memory, random access memory, flash memory) or hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, processor, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for software programming at any time. All or part of the software may sometimes communicate over the internet or other various telecommunications networks. For example, such communications may load software from one computer or processor into another computer or processor, e.g., from a management server or host computer into a computer platform of an application server. Accordingly, another type of medium that may carry software elements includes light waves, electric waves, and electromagnetic waves, such as those used by physical interfaces between local devices, through wired and optical landline networks, and through various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., may also be considered as media carrying software. As used herein, unless limited to a non-transitory tangible "storage" medium, terms, such as computer or machine "readable medium," refer to any medium that participates in providing instructions to a processor for execution.

Thus, a machine-readable medium such as computer-executable code may take many forms, including but not limited to, tangible storage media, carrier wave media, or physical transmission media. As a nonvolatile storage medium, for example, an optical disk or a magnetic disk such as any storage device in any computer, as may be used to implement a database or the like shown in the drawings. Volatile storage media include dynamic liquid storage such as the main memory of a computer platform. Tangible transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electrical or electromagnetic signals, and acoustic or light waves, such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, RAM, ROM, PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium from which a computer can read program code, data, or any combination thereof. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

Computer system 1301 can include or be in communication with an electronic display 1335, electronic display 1335 including a User Interface (UI) 1340 for providing information related to, for example, droplet manipulation, sample manipulation, or a combination thereof. Examples of UIs include, but are not limited to, graphical User Interfaces (GUIs) and web-based user interfaces.

The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithm may be implemented by software executed by the central processor 1105, for example, the algorithm may provide additional liquid to the droplets, replace evaporated solvent of the droplets, map paths of the droplets, or any combination thereof.

Video, input and control of the system may be accessed through a network-based software application. The user input through the software may include images such as drop movement, drop size, and array, and the user input may be recorded and stored in the cloud-based computing system. The stored user input information may be accessed and retrieved in a subset or whole form to provide information for machine learning based algorithms. Drop movement patterns may be recorded and analyzed for training navigation algorithms. The trained algorithm can be used for automation of droplet motion. Spatial fluid characteristics may be recorded and analyzed for training scheme optimization and generation algorithms. The trained algorithms can be used to optimize biological and droplet motion schemes, or to generate new biological and droplet motion schemes. Biological quality control techniques (e.g., amplification-based quantification methods, fluorescence-based quantification methods, absorbance-based quantification methods, surface plasmon resonance methods, and capillary electrophoresis methods for analyzing nucleic acid fragment sizes) can be used to analyze the effectiveness of a workflow performed on an array. The data resulting from these techniques may then be used as input information for a machine learning algorithm to improve the output results. The process may be automated so that the system may iteratively improve the output results.

Examples

Example 1: electrowetting without a dedicated reference electrode

In single-sided electrowetting systems (e.g., the devices and systems described herein), the conductive top plate does not serve as a current return path for the droplets. Instead, dedicated coplanar (or approximately coplanar) electrodes are typically used in combination with actively driven electrode pads. This is to provide a low impedance discharge path for the charge accumulated in the droplet. These electrodes typically take the shape of a mesh grid, with the spacing being the same as the underlying active electrode mesh.

The fabrication and implementation of these coplanar (or nearly coplanar) reference electrodes can complicate electrowetting systems and methods of using such systems. Achieving a low impedance connection with a droplet without disrupting the fluid mobility of the droplet on the surface can be a significant challenge. In contrast, the systems and methods presented herein eliminate the need for a dedicated reference electrode by using adjacent electrodes as current return paths. These systems and methods provide comparable electrowetting properties and completely eliminate manufacturing difficulties associated with the integration of dedicated reference electrodes.

The circuit with a conventional dedicated reference electrode includes a resistive return path for grounding the droplet. Without a dedicated reference electrode, the return path includes a capacitive element formed between one or more inactive electrodes and the droplet sandwiching the dielectric film (fig. 15). For this reason, in order to make this current return path effective, the electrodes need to be activated with a time-varying voltage. The time-varying voltage may be bipolar, in which case the high voltage signal is both positive and negative with respect to the "0V" inactive electrode. In another embodiment, the time-varying voltage may be unipolar, in which case the high voltage signal may only be a positive voltage, and the adjacent electrodes are driven antagonistically such that the electric field across the droplet flip direction is periodically flipped.

The circuit can be driven over a wide frequency range. The lower limit is determined by the hydrodynamic response of the droplet to the stimulus, typically at most 100Hz for droplets in the range of 100nL to 100 μL (but may include the volumes described herein). The upper limit of the frequency range is determined by the RC time constant of the circuit, in fact limited to-1 kHz due to the current limiting resistance in the conductive path. The frequency range can be extended by using dedicated high-voltage circuits supporting higher currents (e.g. up to 20 kHz).

Example 2: electrowetting array comprising a lubricating fluid arranged at the surface of the array

The smooth dielectric film (non-textured) together with the lubricating oil film provides a low friction surface that can be used to effectively manipulate droplets using electrowetting or other digital microfluidic or droplet manipulation methods. In the previous publication (US 20190262829 A1), a thin film of lubricating oil is formed on a textured surface. However, an alternative approach is presented herein that does not require a porous dielectric film to hold the lubricating oil, but rather relies on chemical affinity between the film surface and the lubricating oil.

Similar to the devices and systems described herein, wherein the droplets move on the liquid surface of the textured surface configuration, in the case of an untextured surface, the droplets are again above the surface of the lubricating film. The lubricating film includes a lubricating fluid that is immiscible with the droplets. The lubricating film is thermodynamically stable so that the surface of the dielectric is preferentially wetted and the droplets are on top of the lubricating film. Achieving this stability is important and depends on the affinity of the lubricating liquid for the dielectric surface. For fluorinated surfaces of dielectrics, it may be advantageous to use fluorinated lubricating liquids. Similar chemical structures create greater affinity and lubricants are more likely to wet the surface in a stable manner. On the other hand, when using dielectrics with hydrocarbon-based or siliconized surfaces, such as silicones and untreated polymer plastics, it may be advantageous to use hydrocarbon-based lubricating liquids, such as silicone oils.

Lubricating films may include, but are not limited to:

silicone oil: polydimethyl siloxane, polymethylhydrosiloxane/hydrogen silicone oil, amino silicone oil, phenyl methyl silicone oil, diphenyl silicone oil, vinyl silicone oil, hydroxyl silicone oil, cyclosiloxane and polyoxyalkylene siloxane.

Fluorinated oil: perfluoropolyethers (PFPE), perfluoroalkanes, fluorinated ionic liquids, fluorinated silicone oils, perfluoroalkyl ethers, perfluoro-tri-n-butylamine (FC-40), hydrofluoroether (HFE) liquids.

Other lubricants: ionic liquids, mineral oils, ferrofluids, polyphenylene oxides, vegetable oils, esters of saturated fatty acids and dibasic acids, fats, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils.

The lubricating liquid may contain other functional additives including surfactants, electrolytes, rheology modifiers, waxes, graphite, graphene, molybdenum disulfide, PTFE particles.

Example 3: electrowetting array comprising a filling liquid under a dielectric

The devices and systems described herein generally include a dielectric layer disposed over a coplanar (or approximately coplanar) electrode layer. Described herein are further embodiments wherein the devices and systems include a fill fluid disposed below the dielectric layer.

The fill fluid under the film serves to hold the dielectric film in intimate contact with the underlying PCB substrate and electrode grid by surface tension. When a dielectric film is applied to the surface of the electrode array, the air gap between the film and the electrodes and the air gap between the electrodes is filled with a layer of oil. Thus, the oil layer makes the film adhere to the surface by surface tension while filling the air gap between the electrode and the film. In addition, the oil acts as a high dielectric breakdown material and prevents air breakdown when filling the air gap between any two adjacent electrodes.

Air typically has a breakdown voltage of about 1 kilovolt per millimeter. Thus, while reducing the gap between two adjacent electrodes is advantageous in achieving a smooth transition of the droplet, if the gap between the two electrodes is reduced at some point in time, conduction begins and the electrowetting device fails to function properly. By adding oil to fill the gap between the two electrodes, the gap between the electrodes will be reduced and reliable droplet motion can be achieved with high voltages.

A layer of oil under the dielectric film also has the benefit of smoothing the surface of the film. The dielectric film can be easily stretched and relaxed over the lubricating layer. This property of easy stretching and relaxation makes the film stable and no wrinkles are generated in the film. When wrinkles are created in the film, movement of the droplets may be hindered and/or movement of the droplets may be further hindered. Finally, having a layer of oil under the dielectric film provides a way to easily attach and detach the membrane to and from the electrode array. The filled oil layer here acts as a semi-permanent binder and holds the dielectric layer on the electrode array when the device is in use. However, since it is not permanently fixed to the surface of the electrode array, the user can easily remove the dielectric layer from the surface. Alternatives to not using oil means that the dielectric film/layer is permanently attached to the electrode array or that the user needs more sophisticated instrumentation to uniformly spread the film over the entire surface. In these embodiments, the use of a fill fluid on the electrode array may provide the device and system with a removable "cartridge" that may include a surface to support the droplet.

Example 4: enzyme-catalyzed DNA synthesis

Methods of synthesizing polynucleotides (e.g., DNA) on the arrays described herein using enzyme-catalyzed methods in aqueous media are practiced. Terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase that catalyzes the formation of a phosphodiester bond between the 3 'and 5' ends of DNA. Fig. 5A and 5B illustrate an example workflow for performing DNA synthesis. FIG. 5C shows a schematic of a single reaction site at which stepwise addition of nucleotides is performed to synthesize long molecules of DNA.

Some aspects of the disclosure provide an agent, including an enzyme that mediates synthesis or polymerization. In some embodiments, the first agent, the second agent, the third agent, or any combination thereof, comprises an enzyme that mediates synthesis or polymerization. In some embodiments, the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family.

A droplet containing starting DNA material with unprotected 3' -hydroxyl groups is mixed with a droplet containing functionalized magnetic beads. After a short agitation, the DNA molecules were bound to the magnetic beads. Alternatively, droplets containing starting DNA material are dispensed onto one location of an array that is functionalized to immobilize DNA on a solid support. A droplet containing nucleoside 5' -triphosphate having a cleavable/removable chemical structure is mixed with a droplet containing immobilized starting DNA. The TdT enzyme catalyzes a 5 'to 3' phosphodiester linkage between the unprotected 3 '-hydroxyl end of the starting DNA and the 5' -phosphate end of the nucleoside triphosphate, and the TdT enzyme in the droplets is then combined with the droplets containing the immobilized DNA. The reaction is incubated at room temperature or higher for 5-30 minutes.

Subsequently, the droplets containing the deblocking agent are mixed with a subsequent reaction mixture to produce nucleotides having free 3' -hydroxyl groups. In the case of immobilization using magnetic beads, a magnetic field is then applied to pull the beads down to the surface of the array and remove redundant liquid. Subsequently, the beads are washed a plurality of times (e.g., 2-4 times) by flowing wash buffer over the beads. The washed liquid is then discarded to the waste area of the array. By repeating the above method, additional nucleotides are added to the DNA. During each addition of nucleoside triphosphates, the controller instructs the array to dispense one nucleoside triphosphate from the respective reservoir. After multiple repetitions, polynucleotides of known sequence are produced and remain immobilized on the beads or on the functional surface of the array. By introducing droplets containing a cleavage agent, the final DNA product is cleaved and released from the surface (e.g., the surface of a bead or array). The final product is then suspended in droplets and recovered from the array.

Errors in DNA synthesis can be corrected with mismatch binding proteins and mismatch cleavage proteins. A mismatch binding protein (e.g., mutS) is bound to the magnetic beads and mixed with a droplet containing assembled DNA that contains at least one error (e.g., identified as a double helical twist). For example, DNA molecules containing an error are bound to magnetic beads, while DNA that is free of errors does not adhere to the magnetic beads. The beads are then moved to another area of the array using a magnetic field to remove DNA containing at least one error. Redundant liquids containing error-free DNA are separated from the beads using electrodynamic forces (e.g., EWOD).

Another is to use a mismatch cleaving enzyme, such as T4 endonuclease VII or T7 endonuclease I, to correct the error. The droplets containing the cleaving enzyme are mixed with the droplets containing the assembled DNA. Mismatch cleavage enzymes target the wrong region or regions near the error. The magnetic bead-based separation method is then used to recover the error-free fragments. Alternatively, exonucleases are used to remove other errors from the fragments left by the mismatch cleaving enzyme. These modified fragments were assembled correctly in droplets using PCR assembly.

The assembled and error-corrected DNA was amplified in droplets using PCR. The PCR-derived final product was then prepared into a library for sequencing on an array using the methods described herein. The library was sequenced using any of the sequencing techniques described herein to perform final sequence verification on the synthesized DNA.

The protocol described herein is implemented using a platform employing 10 base initiator DNA. Two synthesis reactions were performed to add 5 and 10 thymine bases to the base DNA. The results were analyzed using denaturing polyacrylamide gel electrophoresis on Azure 600 under blue light irradiation. DNA was coupled to fluorescein dye for visualization. Synthesis was confirmed by agarose gel assay.

The protocols described herein can also be used to prepare functional DNA primers for PCR amplification. Forward and reverse primers were synthesized on a 200pmole scale and required manual post-synthesis treatment. The functionality of the primers was evaluated by performing a 40-cycle PCR protocol and analyzing the results with a 2% agarose gel (e-gel EX). The results confirmed using gel experiments indicate that primers synthesized using the systems and methods described herein have a function comparable to IDT DNA primers based on end-point PCR analysis.

Example 5: extraction of High Molecular Weight (HMW) nucleic acids

Cells from various sources (e.g., mammalian, bacterial, plant) are lysed directly on the array by combining a droplet containing the cell with another droplet containing a lysing agent (e.g., detergent or enzyme). The mixture is heated and mixed (e.g., separately or simultaneously) on an EWOD array to facilitate lysis of the cells and, if applicable, nuclei. Enzymatic digestion of proteins, RNAs, or combinations thereof is performed to increase the purity of the sample. During cell lysis, the progress of the lysis reaction and the lysis efficiency were monitored by DNA specific fluorescent staining. The DNA is purified directly on the array by solid phase (e.g., bead-based capture or by precipitation (e.g., salt and ethanol or phenol-chloroform extraction)). The recovered DNA was handled by EWOD with minimal shear force and transferred to different locations of the array. DNA purity is critical for high quality long read sequencing and can be improved by increasing the number of wash cycles performed on the array. Small DNA fragments were removed using a silicon nanostructured disk. By performing additional sequential elution in buffer, the yield of recovered DNA is increased.

After DNA extraction, samples were analyzed by Pulsed Field Gel Electrophoresis (PFGE), the size distribution of each sample relative to each other was quantified, and analyzed using commercially available standards (BioRad) and ImageJ (NIH) pattern analysis tools. Recovery/size distribution was measured by femtosecond pulses (agilent) for smaller inputs (e.g., cell inputs), and qPCR for lower inputs. Genome integrity was assessed by other complementary methods, such as the BioNano Genomics Saphyr system, allowing for rapid and cost-effective orthographic design on a macroscopic scale, and independent comparison of data using the Saphyr system.

Passivation of EWOD surfaces was determined by testing DNA deposition and retention in the presence of solutions and surface deposited PEG200 or BlockAid (Invitrogen) dulling devices. The assay was done by i) staining the surface after use with Hoechst 33342, ii) calculating the surface retention of the lambda DNA commercial formulation (New England Biolabs, linearized 48.5 kb), and/or iii) measuring the samples before and after the procedure and the% loss of DNA input of 109 to 102 copies by qPCR.

Mammalian cell lysis, RNA and protein digestion were performed on an EWOD array followed by HMW DNA isolation. The distribution of the high molecular weight DNA fragments is shown in FIG. 4, and DNA fragments of more than 165,000bp are isolated from the sample. Longer elution times recover more DNA (e.g., as shown by higher peaks), which is one way to obtain higher DNA yields.

These techniques are used to prepare DNA libraries for sequencing, as described in the examples below.

Example 6: next Generation Sequencing (NGS) library preparation:

the starting material was 224 nanograms (ng) of purified genomic DNA, the DNA source was Genome In A Bottle NA12878. The final library was amplified by two cycles of PCR, which was performed in separate post-PCR zones on a thermocycler. For data comparison, control libraries were performed off-chip manually. The library was quantified with Qubit and the fragment size distribution was assessed using a BioAnalyzer. The library was normalized accordingly and sequenced on NextSeq500 (e.g., shallow sequencing with 2 x 8 cycles for initial mid-output run and index of 2 x 75 cycles followed by additional overlay generation with 2 x 150 cycles of run high output). Sequencing data was demultiplexed using bcl2fastq v2.20 of Illumina without aptamer trimming. Bioinformatics analysis was performed using mature algorithms (e.g., FASTQC, BWA-MEM, SAMtools, picard, and GATK).

The library prepared on the chip produced enough sequencing material (table 1). DNA material generated by the off-chip control is 2.3 times that of the on-chip experiment; however, the average fragment size of both on-chip and off-chip libraries was higher than previously described (table 1 and fig. 6). All sequencing and mapping QC data showed that high quality sequencing libraries were generated using the systems and methods described herein (table 1), with Q30>90% (fig. 7) and%pf reads >90%.

TABLE 1

The level of repetition was low for both on-chip and off-chip libraries (fig. 8), overall below 10% (table 1). The low level of repeated reads is also reflected in the limited content of aptamers. Our initial shallow sequencing (2×75) indicated less than 1% aptamer contamination (fig. 9A), whereas when sequencing depth and read length increased to 2×150, up to 15% and 10% aptamers were detected for the on-chip and off-chip libraries, respectively (fig. 9B). The difference between on-chip and off-chip may be due to the greater number of reads generated by the on-chip library than the off-chip control.

The mapping rate of reads by filtration was high (> 99%) and the genome coverage between the two libraries was comparable (fig. 10), with the median coverage of on-chip and off-chip libraries being 9-fold and 7-fold, respectively. Variation and the ability to invoke Single Nucleotide Polymorphisms (SNPs) were determined. Heterozygous (HET) Single Nucleotide Polymorphism (SNP) sensitivity has the same degree of coverage on-chip and off-chip (table 1). This was confirmed by specifically observing the SNP at the TP53 locus, and detecting the same genotype variation in both libraries of intergenic regions (FIG. 11).

Library preparation based on LSK-110 ligation was performed on a platform to generate a library for analysis on Oxford Nanopore MinION. The kit consumables are prepared and loaded onto the platform along with the film consumables for the platform itself. mu.L of HMW gDNA derived from GM12878 cells was then loaded onto the platform. The system auto-preparation protocol is loaded and started. At the end of the automated protocol, the library was attached to the beads and manually eluted with a magnetic rack after incubation at 37 ℃ for 10 minutes. 12 μl of the supernatant containing the prepared library was transferred to an Oxford Nanopore system for analysis. The results of the experiment are shown in table 2, with figures 32A and 32B showing bar graphs representing the read lengths of two different experimental runs.

TABLE 2

As shown in the results in FIG. 27, the yield of the library prepared on the platform was 40% higher than that of the manual preparation. As shown in FIG. 28, the library also has a high relative pore occupancy when compared to the manually prepared samples, thus showing a high degree of compatibility with the MinION sequencing chemistry. As shown in fig. 30, the base call quality score was also highly similar to that obtained from a manually prepared library. As shown in FIG. 29, the sequencing data also indicated that the N50 of gDNA from this platform was 23kb.

Example 7: workflow sample preparation for DNA samples sequenced on an array:

fig. 12 illustrates an example workflow for NGS on an array as described herein. Cells in the droplets are lysed on the array by introducing another droplet comprising a chemical or enzymatic cell lysis reagent. The proteins contained in the droplets are degraded by introducing a degrading enzyme contained in another droplet of the array, and magnetic particles specific for the DNA molecules are introduced into the droplet containing the DNA molecules. The magnetic beads are attached to the surface of the array or the magnetic beads are suspended in droplets. The DNA molecules are separated from cell debris and degraded proteins using the magnetic field of the array (e.g., a movable magnet as described herein). As shown in fig. 36A-36B, the separated DNA attached to the magnetic particles suspended in the solution is separated from the droplets by translating the movable magnet in a plane parallel to the substrate surface. The isolated DNA coated beads were subjected to a magnetic bead washing process. The DNA is introduced into a DNA sequencer located on, adjacent to, or separate from the array. The DNA was sequenced.

Example 8: high molecular weight DNA extraction using vibration assisted mixing

Purpose(s)

In this experiment, the intended purpose was to extract long and clean DNA from biological samples such as blood, mammalian cells and cultured microorganisms. Typically, the objective is to isolate DNA greater than 50kb (50 kilobases) in length and to isolate sufficient nucleic acid material for downstream applications such as DNA sequencing and optical mapping.

The workflow begins with lysing cells in a biological sample (e.g., cells, blood). Lysis is performed by combining two droplets on an electrowetting array, one containing the cells to be lysed and the other containing the lysis reagent. The lysed cells release all substances from the interior, including long DNA fragments, proteins and other cell debris (collectively "cell lysates"). The mixture of cell lysates is generally quite viscous. Viscous fluids do not move under the force of electrowetting forces or the ability to move is severely impaired (e.g., more energy is required to induce).

A typical method of isolating nucleic acid molecules (e.g., DNA, RNA) from such cell lysate mixtures is to use magnetically sensitive functionalized matrices (e.g., beads, discs) having an affinity for binding to the nucleic acid molecules. Thus, even if magnetic beads are added to a mixture containing cell lysate, it is difficult to mix the matrix with the lysate due to the viscosity of the cell lysate and its non-responsiveness to electrowetting. The matrix will remain stationary in the fluid and will not bind sufficiently to the nucleic acid molecules. In most cases, it may be difficult to proceed to the next step of the workflow to complete the isolation of the nucleic acid molecules. This is because it may be difficult to remove excess liquid from the mixture, which is critical for separating all substances from the matrix-bound nucleic acid molecules. The amount of nucleic acid molecules bound to the matrix is typically very low even if excess fluid is separated. As a result, most of the nucleic acid molecules are lost and the percentage of the target sample that is isolated is very low.

The introduction of vibration/acoustic forces into the system may cause the viscous cell lysate to mix with the magnetic matrix (e.g., DNA and beads). This will encourage the majority of the DNA in the lysate to bind to the beads. Once the DNA is bound to the beads, the excess liquid becomes less viscous. The redundant, less viscous fluid can then be easily separated from the beads using the more standard electrowetting methods described herein.

In addition to allowing the DNA in the cell lysate to bind efficiently to the beads, the vibration assisted mixing also allows for efficient elution of the DNA from the beads (removal of the DNA from the beads). This typically occurs when the DNA-bearing beads are suspended in a solution that releases DNA. In this case, the efficiency is measured by how quickly the DNA is eluted and how much DNA bound to the beads is eluted.

Method

750,000 human cells (GM 12878) were placed on an electrowetting array with and without vibration assisted mixing, from which DNA was extracted. These cells were estimated to contain about 4500ng total genomic DNA. The results of the respective tests are shown in table 3. The total DNA recovered by vibration assisted mixing was about 2830ng and about 63%. Whereas in the absence of vibration, the total DNA recovered was 480ng, only about 11%. Vibration assisted mixing can greatly improve the binding and separation effect of DNA compared to the case of mixing by electrowetting alone.

TABLE 3 Table 3

Example 9: removal of DNA in combination with vibration assisted mixing and use of magnetically responsive beads

In applications where nucleic acid molecules (DNA or RNA) are relevant molecules (e.g., next generation DNA sequencing (NGS), protein sequencing, quantitative PCR (qPCR), drop digital PCR (ddPCR), and other molecular biological applications that immobilize DNA, RNA, proteins, and other biomolecules), functionalized matrices can be used: binding molecules of known size, binding to known types of molecules, and generally for separation and removal from other contaminants. Typical uses of the beads, particularly for the removal of nucleic acids from contaminants, are shown in the following figures.

When the cleaning workflow is performed on an electrowetting device, all the processing is done in droplets, in particular as follows:

first, a droplet composed of the relevant nucleic acid is combined with another droplet composed of the functionalized matrix. In this step, the nucleic acid selectively binds to the beads and leaves all contaminants in solution.

Subsequently, the matrix is pulled down to the surface of the electrowetting array by applying a strong local magnetic field. Once the matrix is granulated, electrowetting forces are used to pull the liquid consisting of the contaminants away from the matrix. In addition, the matrix particles on the surface of the electrowetting array may be washed one or more times with a washing liquid, such as ethanol.

Finally, the matrix is suspended by adding a drop of water or other elution buffer under reduced magnetic field. Subsequently, the nucleic acid bound to the beads is released into solution under aqueous conditions. During the three steps described above, the mixture of substances has a direct effect on the amount of nucleic acid recovered at the end of the workflow. In particular, in the first nucleic acid immobilization step, the matrix in the liquid must be thoroughly mixed to bind the majority of the nucleic acid in the solution. Similarly, the amount of nucleic acid eluted in the final step is proportional to the quality of the mix.

On electrowetting devices (e.g., the devices described herein), mixing using only electrowetting movement may not be sufficient to achieve sufficient binding in the mixing step described immediately above, nor sufficient elution as described above. This can result in the unbound nucleic acid being lost during the removal process. While the amount of nucleic acid bound to the matrix is high when vibration assisted mixing is performed on the electrowetting device. Similarly, nucleic acid is eluted from the matrix by vibration assisted mixing, resulting in the elution of a majority of the nucleic acid. As a result, most DNA was recovered by vibration-assisted mixing.

Method

To demonstrate this, a cleaning reaction of DNA with contaminants was performed using SPRI (solid phase reversible immobilization) magnetic beads.

For this workflow, about 2900ng of DNA was used as input and clean-up was performed using the three steps described above. The workflow is performed without vibration assisted mixing and with vibration assisted mixing, respectively. Both reactions were carried out for about 5 minutes. 2444ng of DNA was recovered when the DNA was cleaned by vibration assisted mixing, as shown in Table 4 below. While when there was no vibration, only about 931ng of DNA was recovered. During the cleaning process performed on the electrowetting device, the amount of DNA recovered from the same input material using vibration assisted mixing was 2.5 times higher than in the case without vibration assisted mixing.

TABLE 4 Table 4

Typically vibration assisted mixing

In general, vibration-assisted mixing helps achieve high quality results that are biologically and chemically relevant, which is difficult to achieve based on single-phase wetting. The above examples illustrate the following modifications:

1. recovery of specific molecules;

2. faster processing of samples; and

3. high efficiency in isolating DNA from highly difficult samples such as cell lysates.

In general, vibration assisted mixing can improve the performance of some other biological processes. Some other processes that may benefit from this include any and all enzyme and bead based reactions in next generation sequencing, protein sequencing, PCR, nucleic acid restriction, nucleic acid digestion, nucleic acid amplification, gene editing, molecular cloning, biopolymer synthesis, biopolymer assembly, DNA repair, RNA repair, DNA ligation, DNA error detection, and DNA replication. In particular, vibration-assisted mixing brings the following advantages in these reactions:

1. These processes are accelerated and thus the time to complete the reaction is shortened;

2. facilitating the efficient use of these enzymes;

3. the input sample consumption is reduced; and

4. the reaction was performed with minimal error.

Furthermore, the technology can be extended to general biological and chemical treatments with liquids containing nucleic acids, proteins, salts, surfactants, beads, cells, metabolites, organic and inorganic molecules.

Example 10: extraction of High Molecular Weight (HMW) genomic DNA (gDNA) from GM12878 cells and whole human blood

Cells from various sources (e.g., mammalian, bacterial, plant) are lysed directly on the array by combining one droplet containing the cells with another droplet containing a lysing agent (e.g., detergent or enzyme). The mixture is heated and mixed (e.g., separately or simultaneously) on an EWOD array to facilitate lysis of the cells and, if applicable, nuclei. Enzymatic digestion of proteins, RNAs, or combinations thereof is performed to increase the purity of the sample. When cells were lysed, progress of the lysis reaction and lysis efficiency were monitored by DNA specific fluorescent staining. The DNA is purified directly on the array using a solid phase (e.g., bead-based capture or by precipitation (e.g., salt and ethanol or phenol-chloroform extraction)). The recovered DNA was passed through EWOD and manipulated with minimal shear force and transferred to different locations on the array. DNA purity is critical to high quality long read sequencing, and can be increased by increasing the number of wash cycles performed on the array. Small DNA fragments can be removed using a silicon nanostructured disk. By performing additional sequential elution in buffer, the yield of recovered DNA is increased.

After DNA extraction, the samples were analyzed by Pulsed Field Gel Electrophoresis (PFGE), the size distribution of each sample relative to each other was quantified, and analyzed using commercially available standards (BioRad) and ImageJ (NIH) pattern analysis tools. Recovery/size distribution was determined by femtosecond pulses (agilent) for smaller inputs (e.g., cell inputs) and qPCR for lower input volumes. Genome integrity is assessed by other complementary methods, such as the Bionano Genomics Saphyr system, enabling rapid and cost-effective prototyping on a macroscopic scale, and independent comparability of data using the Saphyr system.

To obtain isolated HMW DNA from GM12878 cells, the automated extraction process described herein was performed on an EWOD array. The platform performs automated DNA binding, bead spotting, washing and cleaning, and final elution without manual manipulation of the sample. As shown in the results in FIG. 24 and Table 5, more than 5. Mu.g of DNA was extracted within one hour, and had a high purity of more than 100kb in length.

TABLE 5

	GM12878
		Yield (μg)	6.4±0.9
A260/A280	1.84±0.02
		A260/A280	1.93±0.09

The same system was also used to extract HMW gDNA from whole human blood samples. On average, as shown in FIGS. 25A-25C, an average of about 1.2. Mu.g of DNA per 100. Mu.L of whole blood was obtained in each lane within 1 hour. The extraction of a plurality of lanes is performed simultaneously, and as a result gDNA can be isolated rapidly in high yield. PFGE gel analysis was performed to evaluate the extracted fragment length and compared to manual extraction. The results in fig. 26A and 26B show that the extractions performed on the platform have similar or greater average gDNA fragment lengths.

Assays using similar techniques were also successfully performed on a variety of mammalian cell lines including GM06852, GM09237, GM20241, GM07537 and K562.

Example 11: whole genome sequencing of cellular nucleic acids

Genome integrity was demonstrated by long read sequencing with an Oxford Nanopore device. DNA can be extracted using the protocols described herein as well as the Qiagen HMW kit and the Loman protocol. Libraries were prepared according to an optimized protocol to maintain the chains at >1Mb in length. Reproducibility of the extraction was assessed by sequencing a minimum of 3 Qiagen and Loman libraries and 7 Flexomics libraries to ensure robustness of the size performance assessment. Conventional input and low input (e.g., 1000 cells) libraries were evaluated. In the case of low input, 24 subsets were bar coded, 1000 cells per subset, to provide enough material for downstream sequencing (theoretically 150 ng).

For cellular HMW DNA input, titration is performed, for example, by i) supplementing vector DNA, e.g., lambda DNA, to ensure balanced library preparation, or ii) diluting absolute numbers of cells, scaling library preparation and analytical reagents for subsequent reactions. Lambda DNA was biotinylated (e.g., using the Pierce 3' biotinylation kit, thermo Fisher) to be depleted to concentrate the target library prior to sequencing. The performance of ONT transposase library preparation is assessed on the device, e.g., without transferring the sample into a separate tube.

Preparation of samples for whole genome sequencing on various platforms has been demonstrated. HMW gDNA extracted from GM12878 and human whole blood using the extraction protocol described herein, a library was prepared for Illumina NovaSeq 6000. The sequencing data were then compared to manually extracted gDNA. The results in tables 6 and 7 show that the samples extracted by the automated platform have similar quality as the manually prepared samples.

TABLE 6

TABLE 7

To demonstrate the consistency and reliability of library preparation, these preparations and experiments were repeated again using the same inputs and protocols. The results in table 8 show that libraries prepared for Illumina sequencers on the EWOD platform practically exhibit high quality scores on various gDNA sources.

TABLE 8

To demonstrate the consistency of the simplified single droplet reaction automation workflow, the library preparation process was repeated on the Illumina sequencing system. The sizes of the inserts and library fragments were analyzed using Agilent tape station and the results are shown in Table 9. FIGS. 31A and 31B show the library fragment size distribution of one GM12878 and one whole blood sample, respectively.

TABLE 9

To confirm functionality using qPCR-based library quantification, the function of fragments generated in these workflows was also analyzed. The data for these experiments are shown in table 10.

Table 10

Libraries for pacbrio sequence II sequencer were also prepared using an automated platform for gDNA extraction from SMRTcell of GM07537 cell line. The preparation of the library for sequencing on a pacbrio device was performed using the PacBio SMRTbell V3.0.0 kit. A solution of fresh nuclease-free 80% ethanol and 35% AMpure PB beads in elution buffer was prepared. The two solutions were placed in a dispenser platform with nuclease free water. The remaining reagents were calculated as the required volumes for each sample and pipetted into the kit according to the volumes listed in table 11.

TABLE 11

Reagent(s)	Volume in each sample (μl)
		Repair buffer solution	8
Terminal repair mixture	4
		DNA repair mixture	2
SMRT bell aptamer	4
		Connection mixture	30
Ligation enhancers	1
		Nuclease buffer	5
Nuclease mixtures	5
		SMRTbell clear beads	95
Elution buffer	60

Second, the Volta consumable is loaded into the platform by pressing the "load consumable" button of the user interface. When the system prompts, the film consumable is loaded. Subsequently, 46. Mu.L of HMW gDNA was pipetted onto the platform using a wide-bore pipette. The HWM gDNA has a mass of between 300ng and 5 μg and a length of between 15k and 18k base pairs. A260/280 is 1.8, and A260/230 is between 2.0 and 2.2.

After the platform is fully prepared, the appropriate library preparation protocol is loaded by the user. Once the solution is executed, the device performs the entire automated workflow without user intervention. After the protocol was completed, 1.5. Mu.L of supernatant containing the purified library was aspirated with a wide-bore pipette tip and dispensed into 1.5mL LoBind tubes. Subsequently, 1 μl of library was diluted with 9 μl of elution buffer and analyzed using Qubit and femtosecond pulses to determine the quality of the purified library. After confirming that the samples successfully passed the quality management check, gDNA was sequenced using PacBio sequencing equipment. These methods are performed in droplet form on an array using the methods described in this disclosure.

The results of the experiment are shown in fig. 33A to 33E. The raw sequence output produced 512Gb of data, which is higher than the average output of about 300-400Gb experienced by the laboratory performing the experiment. Samples were also subjected to circular consensus sequencing, yielding 32.5Gb HiFi data relative to the expected average 16-20Gb HiFi data. These results demonstrate a 10-fold coverage of the genome of a single SMRTcell, confirming the high quality of extraction in terms of integrity and purity and high compatibility with PacBio sequencing chemistry.

Example 12: preparation of a next Generation library Using QIAseq FX DNA kit

The preparation of the library for sequencing on a QIAseq device was performed using the QIAseq FX DNA library kit. An 80% ethanol solution of fresh nuclease-free water was prepared. This solution was placed into the dispenser platform along with nuclease free water, AMpure XP beads. The remaining reagents were calculated for the required volumes based on the required amounts for each sample and pipetted into the kit according to the volumes listed in table 12.

Table 12

Subsequently, the Volta consumable is loaded into the platform by pressing the "load consumable" button of the user interface. When the system prompts, the film consumable is loaded. Subsequently, 35. Mu.L of HMW gDNA was pipetted into the platform using a 200. Mu.L pipette. HWM gDNA has a mass of between 100ng and 1 μg, A260/280 of 1.8, A260/230 of between 2.0 and 2.2.

After the platform is fully prepared, the appropriate library preparation protocol is loaded by the user and the desired fragmentation time is selected. Once activated, the instrument performs the entire automated workflow without user intervention. After the protocol run was completed, 30. Mu.L of supernatant containing the purified library was aspirated with a pipette tip and dispensed into 1.5ml LoBind tubes. The library was then analyzed for 1 μl with Qubit to determine the quality of the purified library. After confirming that the samples successfully passed the quality management check, gDNA was sequenced again using PacBio sequencing equipment. These methods are performed in droplet form on an array using the methods described in this disclosure.

Example 13: efficiency improvement and cost reduction from continuous platform use

Reliable instrumentation improves the experimental capabilities of the workflow. This accelerates the experimental speed and improves the robustness of the workflow. Figure 35A shows a cumulative experiment running on a platform. The dots within the grey boxes represent individual experiments for automating applications with acceptable bio-output, while the outer dots are internal verification or externally conducted experiments. As shown in fig. 35B, this shows that as platform experience increases, the investment required to develop a new automated workflow, as measured by FTE months, gradually decreases over time. As more test projects develop, efficiency will also prove to increase further.

As the user experience of the platform increases, the quality of the platform bio-output will also increase. Fig. 34 shows the case where the average concentration of DNA extracted using the platform increases with the passage of time. The purity of the extracted DNA also showed an increase at the same time. As the time of use of the platform increases and the number of people using the platform increases, the quality of DNA extracted by the platform and the output of other applications will further increase.

The design of the user test creation software will help to increase the speed and efficiency of future test development. The user can easily change the scheme and customize each program according to his personal application with little knowledge of programming, software or hardware operation. The user can also use a database to store the created solutions, facilitating collaborative development and further improving development speed. This will become clearer as the result of distributing platforms more widely to more users and developing solutions in the platforms for more applications.

The use of machine learning algorithms derived from analysis of platform operation will also improve platform usage efficiency and achieve cost savings over time. These algorithms will help detect and isolate errors that occur during the course of the test. The algorithm then benefits all other users of the same scheme from machine learning based on all users running the scheme. This will greatly improve the development and sophistication of the solution compared to instruments that do not allow for this type of cumulative learning and modification.

Example 14: software architecture

The user interface allows a user to configure actuation of the droplets on the array device (actuation refers to moving, mixing, heating, or other operations of the droplets), which interface may be applied to a computer processor configured to direct the methods and systems described herein. On the user interface, a biological or chemical scheme to be performed on the array device may be defined. Through this interface, liquid related information (such as prescription volume) to be used in the protocol may be entered manually by a user or automatically using natural language processing algorithms. The volume of the prescription can be converted to a volume suitable for the array device (a suitable volume on the array device). This conversion can be achieved by normalizing the maximum and minimum values, followed by calculation of the relative intermediate volume. Liquids with different chemistries exhibit different diffusion behavior across the array device and therefore may occupy different numbers of drive electrodes across the array device. These drop volumes can be adjusted to increase mobility on the array device over a normalized range.

The software interface stores a set of values called "droplet interaction properties". These characteristics may include, but are not limited to, reagent compatibility (the ability of a reagent to contact without affecting biological characteristics), a history of droplet temperature over time, a history of droplet volume, or reagent concentration. The droplet interaction properties may be entered manually by a user or recorded automatically by software using sensors such as temperature probes and optical sensors. These properties may be used to indicate which droplets may contact the same area on the array device. These interaction properties can also be used to determine the ability and order of droplets to contact each other (mix or traverse the same path). The droplets may be grouped by a common attribute in the software to generate a user interface and an automated droplet path. A protocol may be generated by adding droplets to an array device. The automatically calculated volumes may be used to determine the drop footprint on the grid area. These blots can be used to determine the area contaminated with droplets. The contaminated area may be stored and displayed to a user to determine the drop locations on the array device and to clean the available area. During these reactions, the software may direct the evaporation and humidity control methods and systems to the array to keep the physical properties of the droplets, the array itself, and the areas adjacent to the array and/or droplets unchanged.

When a protocol is executed on the device, the "droplet interaction properties" may be recorded. These attributes include, but are not limited to, composition reagents, temperature, presence of sample, and errors during protocol execution. These properties may be displayed on a real-time video feed of droplets on the array device or accessed through simulation of the scheme during execution. The area previously covered by the selected drop may be projected (by a projector mounted above the array device) onto the video feed into the analog grid area or highlighted on the physical grid area.

Data concerning the operation and performance of the device (array device or instrument using the array device) may be collected by various sensors and software components. These sensors may include, but are not limited to, optical sensors, capacitive sensors, temperature sensors, and humidity sensors. Software components may include, but are not limited to, wireless communications, wired communications, device connections, and user interactions. The collected data may be recorded to diagnose operation and failure of the device. The data may also be used to detect errors in real time. These detections may be used to inform the user in real time when user intervention is required. Such intervention may be managed locally by controls available on the device (e.g., physical buttons or software UI elements) or remotely by the user or support team. The collected data may also be used to optimize the user interface.

The digital projector may be mounted on the grid area. The projector may be used to assist a user in manually pipetting liquid into the grid area. This may be accomplished by projection lines or other patterns to guide the user to the desired location or area. During operation, information about drop positions, volumes, and other drop properties can be projected onto the grid area to assist the user in monitoring the scheme. User assistance information may also be displayed when interacting with the device, such as progressing to a desired volume during pipetting. Colors may be projected onto the drops to highlight locations on the array, contaminated areas (areas that have been traversed by another drop), and future paths, thereby associating physical grid areas with software simulations.

The neural network may be trained to test for the presence or absence of droplets in the image. These machine learning models may be trained for various fields of view on a grid area of an array device. These models can then be used to determine which electrode areas are in contact with the droplet. Confidence in drop position may be assigned using an algorithm such as a sliding window method, and then the drop position may be correlated to an expected drop position based on the drop position specified by the programming scheme. This data can be used to adjust the electrode state and flag potential errors in operation. The neural network may also be trained to correlate images of the droplets with their volumes. These models can be created for various types of liquids in order to accurately predict drop volumes with different characteristics. This data can be used to control feedback used in applications such as droplet evaporation. These models can be used for real-time video feeding of droplets during operation of the device. These models can also be used to quickly refine the trial by compiling the learning images into a database for neural network analysis.

The biological protocol file defines the physical manipulations such as liquid mixing and heating, as well as the required reagent and liquid volumes. These schemes are illustrated in terms of separate steps, which include parameters defining the reagents and operations, such as reagent concentration and mixing speed. The feasibility of these operations and fluids on the array device may be determined by examining the parameters, and comparing known limitations of the array device and deducing compatibility. Compatibility of these properties, including but not limited to reagents, physical manipulations, droplet volumes, and chemical reactions, can be determined experimentally. These attributes can then be used to develop a filter for determining whether the standard solution is compatible with the array device. A list of descriptors for these compatible and incompatible attributes can then be compiled and used to create a natural language processing model. The model may be trained to extract the overall structure and the compatibility attributes described above from the standard schema document. The extracted information may be passed through a filter to determine whether the standard solution is compatible with the device. Once compatibility is determined, the critical information may be used to inform the conversion of standard solution operation to device-specific operation. These operations may be compiled and used to generate a device compatible solution. In addition, web search algorithms can be developed that can locate biological protocol documents and compile the documents into a database. The data in the database may then be fed as input to the natural language processing model to determine compatibility and converted to a device schema. These solutions can then be collected and added to a library of solutions.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not limited by the specific examples provided in this specification. While the invention has been described with reference to the above description, the description and illustrations of the embodiments herein are not meant to be a limiting explanation of the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions described herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, the present invention is intended to cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention and the methods and structures within the scope of these claims and their equivalents are covered thereby.

Presently preferred embodiments

Embodiment 1

1. A system for processing a sample, comprising:

a. an array, the array comprising a plurality of electrodes, and a surface configured to support the sample;

b. an electromechanical actuator coupled to the array, wherein the actuator is configured to vibrate the array; and

c. a controller operably coupled to the plurality of electrodes or the electromechanical actuator, wherein the controller is configured to (i) instruct at least a subset of the plurality of electrodes to provide an electric field to alter a wetting characteristic of the surface, or (ii) instruct the electromechanical actuator to apply a vibration frequency to the array.

2. The system of any one of the preceding paragraphs, wherein the controller is configured to perform (i) and (ii).

3. The system of any one of the preceding paragraphs, wherein the controller is coupled to the plurality of electrodes and the electromechanical actuator.

4. The system of any one of the preceding paragraphs, wherein the sample is a droplet.

5. The system of any one of the preceding paragraphs, wherein the droplet comprises about 1 nanoliter to 1 milliliter.

6. The system of any one of the preceding paragraphs, wherein the droplet comprises a biological material.

7. The system of any one of the preceding paragraphs, wherein the biological sample comprises one or more biomolecules.

8. The system of any one of the preceding paragraphs, wherein the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof.

9. The system of any one of the preceding paragraphs, wherein the electrical actuator comprises a cantilever.

10. The system of any one of the preceding paragraphs, wherein the electrical actuator comprises one or more coupling members coupled to the array.

11. The system of any of the preceding paragraphs, wherein the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with a rocking linkage mechanism, or any combination thereof.

12. The system of any of the preceding paragraphs, wherein the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof.

13. The system of any one of the preceding paragraphs, wherein the electromagnetic actuator comprises an electromagnetic voice coil actuator.

14. The system of any one of the preceding paragraphs, wherein the vibration frequency comprises a gradient.

15. The system of any one of the preceding paragraphs, wherein the gradient rises from about a location where the cantilever is coupled to the array.

16. The system of any one of the preceding paragraphs, wherein the vibration has a mode.

17. The system of any one of the preceding paragraphs, wherein the pattern is sinusoidal.

18. The system of any one of the preceding paragraphs, wherein the pattern is square.

19. The system of any one of the preceding paragraphs, wherein the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

20. The system of any one of the preceding paragraphs, wherein the top surface comprises a layer.

21. The system of any one of the preceding paragraphs, wherein the layer comprises a liquid.

22. The system of any one of the preceding paragraphs, wherein the layer comprises a coating.

23. The system of any one of the preceding paragraphs, wherein the coating is hydrophobic.

24. The system of any one of the preceding paragraphs, wherein the layer comprises a film.

25. The system of any one of the preceding paragraphs, wherein the film is a dielectric thin film.

26. The system of any of the preceding paragraphs, wherein the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

27. The system of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

28. The system of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

29. The system of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

30. The system of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

31. The system of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

32. The system of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

33. The system of any of the preceding paragraphs, wherein the first plurality of electrodes, the dielectric, the surface configured to support the droplet containing the sample, or any combination thereof, may be removed from the array.

34. The method of any one of the preceding paragraphs, wherein the electromechanical actuator is configured to displace the surface or a portion of the surface by 0.05 millimeters (mm) to 10mm.

35. The system of any one of the preceding paragraphs, wherein the frequency of the vibration is from 1 hertz (Hz) to 20 kilohertz (kHz).

36. A method for processing a sample, comprising:

a. providing an array, the array comprising:

i. a plurality of electrodes, and

a surface configured to support the sample,

wherein the array is coupled to an electromechanical actuator and the electromechanical actuator is configured to vibrate the array;

b. introducing the droplets to the surface; and

c. the electromechanical actuator is instructed to apply a vibration frequency to the array.

37. The method of any one of the preceding paragraphs, wherein the sample is a droplet.

38. The method of any one of the preceding paragraphs, wherein the droplets comprise about 1 nanoliter to 1 milliliter.

39. The method of any one of the preceding paragraphs, wherein the droplet comprises a biological material.

40. The method of any one of the preceding paragraphs, wherein the biological sample comprises one or more biomolecules.

41. The method of any one of the preceding paragraphs, wherein the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof.

42. The method of any one of the preceding paragraphs, further comprising directing at least a subset of the plurality of electrodes to provide an electric field to alter the wetting characteristics of the surface.

43. The method of any one of the preceding paragraphs, wherein the electromechanical actuator comprises a cantilever.

44. The method of any one of the preceding paragraphs, wherein the electromechanical actuator comprises one or more coupling members coupled to the array.

45. The method of any of the preceding paragraphs, wherein the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with a rocking linkage mechanism, or any combination thereof.

46. The method of any of the preceding paragraphs, wherein the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof.

47. The method of any one of the preceding paragraphs, wherein the electromagnetic actuator comprises an electromagnetic voice coil actuator.

48. The method of any one of the preceding paragraphs, wherein the vibration frequency comprises a gradient.

49. The method of any of the preceding paragraphs, wherein the gradient rises from near a site where the cantilever is coupled to the array.

50. The method of any one of the preceding paragraphs, wherein the vibration has a mode.

51. The method of any one of the preceding paragraphs, wherein the pattern is sinusoidal.

52. The method of any one of the preceding paragraphs, wherein the pattern is square.

53. The method of any one of the preceding paragraphs, wherein the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

54. The method of any one of the preceding paragraphs, wherein the surface comprises a layer disposed on a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

55. The method of any one of the preceding paragraphs, wherein the layer comprises a liquid.

56. The method of any one of the preceding paragraphs, wherein the layer comprises a coating.

57. The method of any one of the preceding paragraphs, wherein the coating is hydrophobic.

58. The method of any one of the preceding paragraphs, wherein the layer comprises a film.

59. The method of any one of the preceding paragraphs, wherein the film is a dielectric film.

60. The method of any of the preceding paragraphs, wherein the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

61. The method of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

62. The method of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

63. The method of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

64. The method of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

65. The method of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, fats, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

66. The method of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, electrolyte, rheology modifier, wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

67. The method of any of the preceding paragraphs, wherein the first plurality of electrodes, the dielectric, the surface configured to support the droplet containing the sample, or any combination thereof, may be removed from the array.

68. The method of any one of the preceding paragraphs, wherein the frequency of the vibration displaces the surface or a portion of the surface by 0.05 millimeters (mm) to 10mm.

69. The method of any one of the preceding paragraphs, wherein the frequency of the vibration is from 1 hertz (Hz) to 20 kilohertz (kHz).

70. A method of contacting a first sample with a second sample, wherein the first sample is contained in a first droplet and the second sample is contained in a second droplet, the method comprising:

a. providing an array, the array comprising:

i. a plurality of electrodes; and

a surface configured to support the first droplet and the second droplet, wherein the array is coupled to an electromechanical actuator, and the electromechanical actuator is configured to vibrate the array;

b. Directing the first droplet and the second droplet to the surface;

c. directing at least a subset of the plurality of electrodes to provide an electric field to change a wetting characteristic of the surface, thereby inducing movement of the first and second droplets, wherein the movement of the first and second droplets comprises the first and second droplets converging to generate a mixed droplet; and

d. instructing the electromechanical actuator to apply a vibration frequency to the surface;

thereby bringing the first sample into contact with the second sample.

71. The method of any one of the preceding paragraphs, wherein the first sample, the second sample, or both comprise a viscous fluid.

72. The method of any one of the preceding paragraphs, wherein the first sample, the second sample, or both comprise a biological sample.

73. The method of any one of the preceding paragraphs, wherein the biological sample comprises one or more biomolecules.

74. The method of any one of the preceding paragraphs, wherein the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof.

75. The method of any one of the preceding paragraphs, wherein the first sample, the second sample, or both comprise a reagent for a biological assay.

76. The method of any one of the preceding paragraphs, wherein the first sample, the second sample, or both comprise one or more cell lysis reagents.

77. The method of any one of the preceding paragraphs, wherein the one or more cell lysis reagents comprise a matrix configured to bind to a biological sample or a subset of the biological sample.

78. The method of any one of the preceding paragraphs, wherein the nucleic acid molecule comprises more than 100 bases, 1 kilobase (kb), 20kb, 30kb, 40kb or 50kb.

79. The method of any one of the preceding paragraphs, wherein more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the biological sample is bound to the matrix.

80. The method of any one of the preceding paragraphs, wherein the substrate is a functionalized bead.

81. The method of any one of the preceding paragraphs, wherein the substrate is a functionalized disc.

82. The method of any of the preceding paragraphs, wherein the method further comprises, after d), performing e) altering the wetting characteristics of the surface by directing at least a subset of the plurality of electrodes to provide an electric field to induce movement in at least the portion of the mixed droplet to thereby remove at least a portion of the mixed droplet.

83. The method of any one of the preceding paragraphs, wherein at least the portion of the mixed droplet does not contain the biological sample.

84. The method of any one of the preceding paragraphs, wherein the method further comprises applying a magnetic field to the surface prior to or simultaneously with (e).

85. The method of any one of the preceding paragraphs, wherein the magnetic field immobilizes the substrate.

86. The method of any one of the preceding paragraphs, wherein the electromechanical actuator comprises a cantilever.

87. The method of any one of the preceding paragraphs, wherein the electromechanical actuator comprises one or more coupling members coupled to the array.

89. The method of any of the preceding paragraphs, wherein the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with a rocking linkage mechanism, or any combination thereof.

90. The method of any of the preceding paragraphs, wherein the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof.

91. The method of any one of the preceding paragraphs, wherein the electromagnetic actuator comprises an electromagnetic voice coil actuator.

92. The method of any one of the preceding paragraphs, wherein the vibration frequency comprises a gradient.

93. The method of any of the preceding paragraphs, wherein the gradient rises from near a site where the cantilever is coupled to the array.

94. The method of any one of the preceding paragraphs, wherein the vibration has a mode.

95. The method of any one of the preceding paragraphs, wherein the pattern is sinusoidal.

96. The method of any one of the preceding paragraphs, wherein the pattern is square.

97. The method of any one of the preceding paragraphs, wherein the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

98. The method of any one of the preceding paragraphs, wherein the surface comprises a layer disposed on a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

99. The method of any one of the preceding paragraphs, wherein the layer comprises a liquid.

100. The method of any one of the preceding paragraphs, wherein the layer comprises a coating.

101. The method of any one of the preceding paragraphs, wherein the coating is hydrophobic.

102. The method of any one of the preceding paragraphs, wherein the layer comprises a film.

103. The method of any one of the preceding paragraphs, wherein the film is a dielectric film.

104. The method of any of the preceding paragraphs, wherein the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

105. The method of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

106. The method of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

107. The method of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

108. The method of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

109. The method of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, fats, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

110. The method of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, electrolyte, rheology modifier, wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

111. The method of any of the preceding paragraphs, wherein the first plurality of electrodes, the dielectric, the surface configured to support the droplet containing the sample, or any combination thereof, may be removed from the array.

112. The method of any one of the preceding paragraphs, wherein the frequency of the vibration displaces the surface or a portion of the surface by 0.05 millimeters (mm) to 10mm.

113. The method of any one of the preceding paragraphs, wherein the frequency of the vibration is from 1 hertz (Hz) to 20 kilohertz (kHz).

114. A system for processing droplets, comprising:

a. an array, the array comprising:

i. a plurality of electrodes, wherein none of the plurality of electrodes is permanently grounded, an

A surface configured to support a droplet comprising the sample;

b. a controller operably coupled to the plurality of electrodes, wherein the controller is configured to:

i. At least a subset of the plurality of electrodes is activated with a time-varying voltage to change the wetting characteristics of the surface.

115. The system of any one of the preceding paragraphs, wherein the system does not include a cover electrode.

116. The system of any of the preceding paragraphs, wherein the plurality of electrodes comprises at least one electrode having a cross-section or overlap with the droplet sufficient to form a current return path in the vicinity of the electrode and adjacent electrodes.

117. The system of any one of the preceding paragraphs, wherein the overlap of the droplet with at least one electrode is sufficient to generate a minimized energy state or equilibrium state in the droplet.

118. The system of any one of the preceding paragraphs, wherein the plurality of electrodes are coplanar.

119. The system of any one of the preceding paragraphs, wherein the time-varying voltage is bipolar.

120. The system of any one of the preceding paragraphs, wherein the time-varying voltage is from about 1Hz to about 20kHz.

121. The system of any of the preceding paragraphs, wherein upon activation of at least the subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and one or more inactive electrodes.

122. The system of any one of the preceding paragraphs, wherein the activation of at least the subset of the plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

123. The system of any one of the preceding paragraphs, further comprising a dielectric layer.

124. The system of any one of the preceding paragraphs, wherein the dielectric layer comprises a thickness, wherein the thickness is sufficient to ground current generated by the plurality of electrodes.

125. The system of any one of the preceding paragraphs, wherein the dielectric layer comprises a thickness, wherein the thickness is sufficient to act as at least a portion of an electrical barrier.

126. The system of any one of the preceding paragraphs, wherein the thickness is from 0.025 micrometers (μm) to 10,000 μm.

127. The system of any of the preceding paragraphs, wherein the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

128. The system of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

129. The system of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

130. The system of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

131. The system of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

132. The system of any one of the preceding paragraphs, wherein the surface comprises a liquid layer.

133. The system of any of the preceding paragraphs, wherein the liquid layer comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

134. The system of any of the preceding paragraphs, wherein the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

135. The system of any one of the preceding paragraphs, further comprising a liquid disposed in the gap adjacent the dielectric layer and the plurality of electrodes.

136. The system of any one of the preceding paragraphs, wherein the liquid creates an adhesion between the plurality of electrodes and the dielectric layer.

137. The system of any one of the preceding paragraphs, wherein the liquid comprises a dielectric material.

138. The system of any of the preceding paragraphs, wherein the liquid prevents or reduces electrical conductivity of air disposed in the gap.

139. The system of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

140. The system of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

141. A system for processing droplets, comprising:

a. an array, the array comprising:

A surface configured to support a droplet comprising the sample;

i. activating at least a subset of the plurality of electrodes with a voltage to change a wetting characteristic of the surface;

wherein the array does not include a permanent reference electrode.

142. The system of any of the preceding paragraphs, wherein the voltage is a time-varying voltage.

143. The system of any one of the preceding paragraphs, wherein the system does not include a cover electrode.

144. The system of any one of the preceding paragraphs, wherein the plurality of electrodes comprises at least one electrode having a cross-section or overlap with the droplet sufficient to create a current return path in the vicinity of the electrode and adjacent electrodes.

145. The system of any one of the preceding paragraphs, wherein said overlapping of said droplet with at least one electrode is sufficient to create a minimized energy state or equilibrium state in said droplet.

146. The system of any one of the preceding paragraphs, wherein the plurality of electrodes are coplanar.

147. The system of any one of the preceding paragraphs, wherein the time-varying voltage is bipolar.

148. The system of any one of the preceding paragraphs, wherein the time-varying voltage is from about 1Hz to about 20kHz.

149. The system of any of the preceding paragraphs, wherein upon activation of at least the subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and one or more inactive electrodes.

150. The system of any one of the preceding paragraphs, wherein the activation of at least the subset of the plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

151. The system of any one of the preceding paragraphs, further comprising a dielectric layer.

152. The system of any one of the preceding paragraphs, wherein the dielectric layer comprises a thickness, wherein the thickness is sufficient to ground current generated by the plurality of electrodes.

153. The system of any one of the preceding paragraphs, wherein the dielectric layer comprises a thickness, wherein the thickness is sufficient to act as at least a portion of an electrical barrier.

154. The system of any one of the preceding paragraphs, wherein the thickness is from 0.025 micrometers (μm) to 10,000 μm.

155. The system of any of the preceding paragraphs, wherein the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

156. The system of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

157. The system of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

158. The system of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

159. The system of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

160. The system of any one of the preceding paragraphs, wherein the surface comprises a liquid layer.

161. The system of any of the preceding paragraphs, wherein the liquid layer comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

162. The system of any of the preceding paragraphs, wherein the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

163. The system of any one of the preceding paragraphs, further comprising a liquid disposed in the gap adjacent the dielectric layer and the plurality of electrodes.

164. The system of any one of the preceding paragraphs, wherein the liquid creates an adhesion between the plurality of electrodes and the dielectric layer.

165. The system of any one of the preceding paragraphs, wherein the liquid comprises a dielectric material.

166. The system of any one of the preceding paragraphs, wherein the liquid prevents or reduces conductivity of air disposed in the gap.

167. The system of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

168. The system of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

169. A method for moving a droplet on an array, wherein the array comprises a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded, and configured to support a surface of the droplet containing the sample, the method comprising:

a) Activating at least a subset of the plurality of electrodes with a time-varying voltage to alter the wetting characteristics of the surface;

wherein the time-varying voltage generates a current return path in the vicinity of the droplet and the one or more inactive electrodes, thereby causing movement of the droplet.

170. The method of any one of the preceding paragraphs, wherein the plurality of electrodes are coplanar.

171. A method as in any preceding paragraph, wherein the time-varying voltage is bipolar.

172. The method of any one of the preceding paragraphs, wherein the time-varying voltage is from about 1Hz to about 20kHz.

173. The method of any of the preceding paragraphs, wherein, upon activating at least the subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and one or more inactive electrodes.

174. The method of any one of the preceding paragraphs, wherein said activating of at least said subset of said plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

175. A method for moving a droplet on an array, wherein the array comprises a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded, and configured to support a surface of the droplet containing the sample, the method comprising:

a. activating at least a subset of the plurality of electrodes with a voltage to change a wetting characteristic of the surface;

wherein the array does not include a permanent reference electrode.

176. A method as in any preceding paragraph, wherein the time-varying voltage generates a current return path in the vicinity of the droplet and one or more inactive electrodes, thereby causing movement of the droplet.

177. The method of any one of the preceding paragraphs, wherein the plurality of electrodes are coplanar.

178. A method as in any preceding paragraph, wherein the time-varying voltage is bipolar.

179. The method of any one of the preceding paragraphs, wherein the time-varying voltage is from about 1Hz to about 20kHz.

180. The method of any of the preceding paragraphs, wherein, upon activating at least the subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and one or more inactive electrodes.

181. The method of any one of the preceding paragraphs, wherein said activating of at least said subset of said plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

182. A system for processing a sample, comprising:

a. a plurality of electrodes;

b. a dielectric layer disposed over the plurality of electrodes, wherein the dielectric layer comprises a surface configured to support a droplet comprising the sample;

c. a liquid disposed in the gap adjacent to the plurality of electrodes and the dielectric layer.

183. The system of any one of the preceding paragraphs, wherein the liquid creates an adhesion between the plurality of electrodes and the dielectric layer.

184. The system of any one of the preceding paragraphs, wherein the liquid comprises a dielectric material.

185. The system of any one of the preceding paragraphs, wherein the liquid prevents or reduces conductivity of air disposed in the gap.

186. The system of any of the preceding paragraphs, wherein the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

187. The system of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

188. The system of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

189. The system of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

190. The system of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

191. The system of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

192. The system of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

193. The system of any one of the preceding paragraphs, wherein the surface comprises a liquid layer.

194. The system of any of the preceding paragraphs, wherein the liquid layer comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

195. The system of any of the preceding paragraphs, wherein the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

196. The system of any one of the preceding paragraphs, wherein the dielectric layer is removable.

197. A system for processing a sample, comprising:

a. a plurality of electrodes;

c. A liquid adjacent to the surface, wherein the liquid has a chemical affinity for the surface, wherein the chemical affinity is sufficient to immobilize the liquid on the surface, and wherein the liquid is resistant to gravity.

198. The system of any of the preceding paragraphs, wherein the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

199. The system of any of the preceding paragraphs, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

200. The system of any of the preceding paragraphs, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

201. The system of any of the preceding paragraphs, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

202. The system of any of the preceding paragraphs, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

203. The system of any of the preceding paragraphs, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

204. The system of any of the preceding paragraphs, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

205. The system of any one of the preceding paragraphs, wherein the surface comprises a liquid layer.

206. The system of any of the preceding paragraphs, wherein the liquid layer comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

207. The system of any of the preceding paragraphs, wherein the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

208. The system of any one of the preceding paragraphs, wherein the dielectric layer is removable.

Embodiment 2

1. A method of producing a biopolymer, comprising:

a. providing a plurality of droplets adjacent to the surface, wherein the plurality of droplets comprises a first droplet comprising a first reagent and a second droplet comprising a second reagent;

b. Moving the first droplet and the second droplet relative to each other to (i) contact the first droplet with the second droplet, and (ii) form a combined droplet comprising the first reagent and the second reagent; and

c. in the combined droplet, at least a portion of the biopolymer is formed using at least (i) the first reagent and (ii) the second reagent, wherein (b) - (c) are performed in 10 minutes or less.

2. The method of paragraph 1 wherein the biopolymer is a polynucleotide.

3. The method of paragraph 1 wherein the biopolymer is a polypeptide.

4. The method of any one of paragraphs 1 or 2, wherein the polynucleotide comprises about 10 to about 250 bases.

5. The method of any one of paragraphs 1 to 3, wherein the polynucleotide comprises about 260 to about 1kb.

6. The method of any one of paragraphs 1 to 3, wherein the polynucleotide comprises about 1kb to about 10,000kb.

7. The method of any one of paragraphs 1 to 6, wherein vibration is applied to the synthetic droplets during b), c), or both.

8. The method of any one of paragraphs 1 to 7, wherein the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets.

9. The method of paragraph 8, wherein vibration is applied to the one or more cleaning steps.

10. The method of any one of paragraphs 1 to 9, wherein the surface is a dielectric.

11. The method of any one of paragraphs 1 to 9, wherein the surface comprises a dielectric layer disposed over the one or more electrodes.

12. The method of any one of paragraphs 1 to 9, wherein the surface is a surface of a polymer film.

13. The method of any one of paragraphs 1 to 9, wherein the surface comprises one or more oligonucleotides bound to the surface.

14. The method of any one of paragraphs 1 to 9, wherein the surface is a surface of a layer of lubricating liquid.

15. The method of any one of paragraphs 1 to 14, wherein the plurality of droplets comprises a third droplet comprising a third reagent.

16. The method of any one of paragraphs 1 to 15, wherein the first agent, the second agent, the third agent, or any combination thereof comprises one or more functionalized beads.

17. The method of paragraph 16, wherein the functionalized bead comprises one or more oligonucleotides immobilized thereto.

18. The method of any one of paragraphs 1 to 17, wherein vibration is applied to the first droplet, the second droplet, the third droplet, the wash droplet, or a mixture thereof.

19. The method of any one of paragraphs 1 to 18, wherein the first agent, the second agent, the third agent, or any combination thereof comprises a polymerase.

20. The method of any one of paragraphs 1 to 19, wherein the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer.

21. The method of paragraph 20 wherein the biomonomer is an amino acid.

22. The method of paragraph 20 wherein the biomonomer is a nucleic acid molecule.

23. The method of paragraph 22, wherein the nucleic acid molecule comprises adenine, cytosine, guanine, thymine, or uracil.

24. The method of any one of paragraphs 1 to 23, wherein the first agent, the second agent, the third agent, or any combination thereof comprises one or more functionalized discs.

25. The method of paragraph 24 wherein the functionalized disc comprises one or more oligonucleotides immobilized thereto.

26. The method of any one of paragraphs 1 to 25, wherein the first agent, the second agent, the third agent, or any combination thereof comprises an enzyme that mediates synthesis or polymerization.

27. The method of paragraph 26, wherein the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family.

28. The method of any one of paragraphs 1 to 27, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 20 minutes or less.

29. The method of any one of paragraphs 1 to 28, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets within 15 minutes or less.

30. The method of any one of paragraphs 1 to 29, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 10 minutes or less.

31. The method of any one of paragraphs 1 to 30, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 1 minute or less.

32. The method of any one of paragraphs 1 to 31, wherein the combined droplets are temperature controlled.

33. The method of any one of paragraphs 1 to 32, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field.

34. The method of any one of paragraphs 1 to 33, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to illumination.

35. The method of any one of paragraphs 1 to 34, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change.

36. The method of any one of paragraphs 1 to 35, wherein the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps).

37. The method of paragraph 36, wherein the deoxynucleoside triphosphate can have a protecting group.

38. The method of paragraph 37 wherein the protecting group may be removed in the reaction.

39. The method of any one of paragraphs 1 to 38, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side.

40. The method of any one of paragraphs 1 to 39, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 nanoliter (1 nL) and 500 microliters (500 μl).

41. The method of any one of paragraphs 1 to 40, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 microliter (1 μl) and 500 microliters (500 μl).

42. The method of any one of paragraphs 1 to 41, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 microliter (1 μl) and 200 microliters (200 μl).

43. The method of any one of paragraphs 1 to 42, wherein the method further comprises attaching the biopolymer to a second biopolymer.

44. The method of paragraph 43, wherein the second biopolymer is produced using the method of any of paragraphs 1 to 43.

45. A method of producing a biopolymer, comprising:

c. forming at least a portion of the biopolymer using at least (i) the first reagent and (ii) the second reagent in the combined droplet;

wherein vibration is applied to (b), (c), or both.

46. The method of paragraph 45, wherein the biopolymer is a polynucleotide.

47. The method of paragraphs 45 or 46, wherein the biopolymer is a polypeptide.

48. The method of any one of paragraphs 45 to 47, wherein the polynucleotide comprises 2 to 10,000,000 nucleic acid molecules.

49. The method of any one of paragraphs 45 to 48, wherein the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets.

50. The method of paragraph 49, wherein vibration is applied to the one or more cleaning steps.

51. The method of any one of paragraphs 45 to 50, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 30 minutes or less.

52. The method of any one of paragraphs 45 to 51, wherein the surface is a dielectric.

53. The method of any one of paragraphs 45 to 51, wherein the surface comprises a dielectric layer disposed over the one or more electrodes.

54. The method of any one of paragraphs 45 to 51, wherein the surface is a surface of a polymer film.

55. The method of any one of paragraphs 45 to 51, wherein the surface comprises one or more oligonucleotides bound to the surface.

56. The method of any one of paragraphs 45 to 51, wherein the surface is a surface of a lubricating liquid layer.

57. The method of any one of paragraphs 45 to 56, wherein the plurality of droplets comprises a third droplet comprising a third reagent.

58. The method of any one of paragraphs 45 to 57, wherein the first reagent, the second reagent, the third reagent, or any combination thereof comprises one or more functionalized beads.

59. The method of any one of paragraphs 45 to 58, wherein the functionalized bead comprises one or more oligonucleotides immobilized thereto.

60. The method of any one of paragraphs 45 to 59, wherein the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase.

61. The method of any one of paragraphs 45 to 60, wherein the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer.

62. The method of paragraph 61 wherein the biomonomer is an amino acid.

63. The method of paragraph 61 wherein the biomonomer is a nucleic acid molecule.

64. The method of paragraph 63, wherein the nucleic acid molecule is adenine, cytosine, guanine, thymine, or uracil.

65. The method of any one of paragraphs 45 to 64, wherein the first agent comprises one or more functionalized discs.

66. The method of any one of paragraphs 45 to 65, wherein the functionalized disc comprises one or more oligonucleotides immobilized thereto.

67. The method of any one of paragraphs 45 to 66, wherein the first droplet, the second droplet, the third droplet, or a combination thereof comprises an enzyme that mediates synthesis or polymerization.

68. The method of paragraph 67, wherein the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family.

69. The method of any one of paragraphs 45 to 68, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets within 20 minutes or less.

70. The method of any one of paragraphs 45 to 69, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 15 minutes or less.

71. The method of any one of paragraphs 45 to 70, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 10 minutes or less.

72. The method of any one of paragraphs 45 to 71, wherein the combined droplets are heated.

73. The method of any one of paragraphs 45 to 71, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field.

74. The method of any one of paragraphs 45 to 71, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to illumination.

75. The method of any one of paragraphs 45 to 71, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change.

76. The method of any one of paragraphs 45 to 75, wherein the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps).

77. The method of paragraph 76, wherein the deoxynucleoside triphosphate can have a protecting group.

78. The method of paragraph 77, wherein the protecting group may be removed in a reaction.

79. The method of any one of paragraphs 45 to 78, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is in contact with a surface on only one side.

80. The method of any one of paragraphs 45 to 79, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 nanoliter (1 nL) and 500 microliters (500 μl).

81. The method of any one of paragraphs 45 to 80, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 microliter (1 μl) and 500 microliters (500 μl).

82. The method of any one of paragraphs 45 to 81, wherein the volume of the first droplet, the second droplet, the third droplet, or the combined droplet is between 1 microliter (1 μl) and 200 microliters (200 μl).

Embodiment 3

1. A method for circularizing a nucleic acid sample, comprising:

a. providing a droplet adjacent to an electrowetting array, wherein the sample droplet comprises the nucleic acid sample; and

b. the droplets are treated using the electrowetting array to circularize the nucleic acid sample.

2. The method of paragraph 1, wherein the electrowetting array comprises a dielectric matrix.

3. The method of paragraph 1, wherein the electrowetting array further comprises one or more reagent droplets.

4. The method of paragraph 1, wherein the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample.

5. The method of paragraph 4, further comprising:

a. combining the sample droplet with the one or more reagent droplets;

b. separating the sample droplets from the one or more reagent droplets; and

c. combining the one or more reagent droplets with a second droplet.

6. The method of paragraph 1, wherein the droplet comprises one or more reagents for circularizing the nucleic acid sample.

7. The method of paragraph 4, wherein (b) further comprises performing one or more droplet operations on the electrowetting array to treat the droplet, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplet.

8. The method of paragraph 3, wherein the electrowetting array comprises one or more electrodes located below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplet, or both.

9. The method of paragraph 7, wherein the one or more droplet operations comprise applying vibration to the one or more reagent droplets, the sample droplet, or both.

10. The method of paragraph 7, wherein the one or more droplet operations comprise applying vibration to the electrowetting array.

11. The method of paragraph 1, further comprising performing a sequencing reaction on the nucleic acid sample using a single polymerase.

12. The method of paragraph 1, further comprising generating a sequencing read that is at least 70 kilobases (kb) in length.

13. The method of paragraph 1, further comprising generating a sequencing read that is at least 80 kilobases (kb) in length.

14. The method of paragraph 1, further comprising generating a sequencing read of about 200 kilobases (kb) in length.

15. The method of paragraph 1, wherein sequencing data of at least 100 (Gb) is generated.

16. The method of paragraph 15 wherein sequencing data of at least 500Gb is generated.

17. The method of paragraph 16, wherein at least 512Gb of sequencing data is generated.

18. The method of paragraph 1 wherein at least 10Gb of data is generated.

19. The method of paragraph 18, wherein at least 30Gb of data is generated.

20. The method of paragraph 11, wherein the sequencing reaction comprises repeated cycle sequencing.

21. The method of paragraph 12 wherein one or more sub-reads of the sequencing reads are generated.

22. The method of paragraph 21 wherein the consensus sequence is generated from the sub-reads of the sequencing reads.

23. The method of paragraph 13, wherein the sequencing read has an A260/A280 ratio of less than about 1.84.

24. The method of paragraph 1, further comprising generating a circularized nucleic acid sample.

25. The method of paragraph 24 wherein the circularized nucleic acid sample comprises a target sequence.

26. The method of paragraph 24, wherein the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence.

27. The method of paragraph 25, wherein at least 80% of the plurality of sequences comprise the target sequence.

28. The method of paragraph 1, wherein the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

29. A method of sequencing a nucleic acid sample comprising (a) providing a droplet adjacent to an electrowetting array, the droplet comprising the nucleic acid sample, (b) treating the droplet with the electrowetting array to circularize the nucleic acid sample, and (c) performing a sequencing reaction on the circularized nucleic acid sample using a single polymerase.

30. A method of sequencing a circular nucleic acid sample comprising performing a sequencing reaction on the nucleic acid sample using a single polymerase to produce a sequencing read of at least 70 kilobases in length.

31. The method of paragraph 29, further comprising using a waveguide to detect bases incorporated into the nucleic acid sample during the sequencing reaction.

32. A method of producing a circularised nucleic acid sample having a longer insert size, comprising (a) providing a droplet adjacent to an electrowetting array, the droplet comprising the nucleic acid sample, (b) treating the droplet with the electrowetting array to circularise the nucleic acid sample, and (c) performing a sequencing reaction on the circularised nucleic acid sample using a single polymerase.

33. A method for generating a sequencing library, comprising (a) providing a nucleic acid sample comprising a plurality of nucleic acid molecules, the nucleic acid molecules comprising a plurality of sequences, and (b) generating the sequencing library using the nucleic acid sample, wherein the sequencing library comprises at least 80% of the plurality of sequences of its complement.

34. A method for circularizing a nucleic acid sample, comprising:

(a) Providing a droplet adjacent to an electrowetting array, wherein the droplet comprises the nucleic acid sample;

(b) Combining the droplets with one or more reagent droplets;

(c) Processing the droplets using the electrowetting array to circularize the nucleic acid sample;

(d) Separating the droplets from the one or more reagent droplets; and

(e) Combining the one or more reagent droplets with the sample droplet to generate a circularized nucleic acid sample.

35. The method of any one of the preceding paragraphs, wherein the electrowetting array comprises a dielectric matrix.

36. The method of any one of the preceding paragraphs, wherein the electrowetting array further comprises one or more reagent droplets.

37. The method of any one of the preceding paragraphs, wherein the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample.

38. The method of any one of the preceding paragraphs, further comprising:

(a) Combining the sample droplet with the one or more reagent droplets;

(b) Separating the sample droplet from the one or more reagent droplets; and

(c) Combining the one or more reagent droplets with a second droplet.

39. The method of any one of the preceding paragraphs, wherein the droplet comprises one or more reagents for circularizing the nucleic acid sample.

40. The method of any one of the preceding paragraphs, wherein b) further comprises performing one or more droplet operations on the electrowetting array to treat the droplet, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplet.

41. The method of any of the preceding paragraphs, wherein the electrowetting array comprises one or more electrodes located below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the reagent droplet, the sample droplet, or both.

42. The method of any one of the preceding paragraphs, wherein the one or more droplet operations comprise applying vibration to the one or more reagent droplets, the sample droplet, or both.

43. A method as in any preceding paragraph, wherein the one or more droplet operations comprise applying vibration to the electrowetting array.

44. The method of any one of the preceding paragraphs, further comprising performing a sequencing reaction on the nucleic acid sample using a single polymerase.

45. The method of any one of the preceding paragraphs, further comprising generating a sequencing read of at least 70 kilobases (kb) in length.

46. The method of any one of the preceding paragraphs, further comprising generating a sequencing read of at least 80 kilobases (kb) in length.

47. The method of any one of the preceding paragraphs, further comprising generating a sequencing read of about 200 kilobases (kb) in length.

48. The method of any one of the preceding paragraphs, wherein sequencing data of at least 100 (Gb) is generated.

49. The method of any one of the preceding paragraphs, wherein sequencing data of at least 500Gb is generated.

50. The method of paragraph 16, wherein at least 512Gb of sequencing data is generated.

51. A method as in any preceding paragraph, wherein at least 10Gb of data is generated.

52. A method as in any preceding paragraph, wherein at least 30Gb of data is generated.

53. The method of any one of the preceding paragraphs, wherein the sequencing reaction comprises repeated cycle sequencing.

54. The method of any one of the preceding paragraphs, wherein one or more sub-reads of the sequencing reads are generated.

55. The method of any one of the preceding paragraphs, wherein the consensus sequence is generated from said sub-reads of the sequencing reads.

56. The method of any one of the preceding paragraphs, wherein the sequencing reads have an a260/a280 ratio of less than about 1.84.

57. The method of any one of the preceding paragraphs, further comprising generating a circularized nucleic acid sample.

58. The method of any one of the preceding paragraphs, wherein the circularized nucleic acid sample comprises a target sequence.

59. The method of any one of the preceding paragraphs, wherein the circularized nucleic acid sample comprises a plurality of sequences, the plurality of sequences comprising the target sequence.

60. The method of any one of the preceding paragraphs, wherein at least 80% of the plurality of sequences comprise the target sequence.

61. The method of any one of the preceding paragraphs, wherein the method further comprises obtaining the nucleic acid sample from a biological sample on the electrowetting array prior to (a).

62. A method for processing a nucleic acid sample, comprising:

(a) Providing a biological sample adjacent to an electrowetting array, wherein the sample droplet comprises the nucleic acid sample; and

(b) Extracting the nucleic acid sample from the biological sample adjacent to the electrowetting array;

wherein the nucleic acid sample comprises a sequencing read that is at least about 70 kilobases (kb) in length.

63. The method of paragraph 61 wherein the length is at least about 80 kilobases (kb).

64. The method of paragraph 61 wherein the length is at least about 200 kilobases (kb).

65. The method of paragraph 61 wherein the sequencing read has an A260/A280 ratio of less than about 1.84.

Claims

1. A method of producing a biopolymer, the method comprising:

c. in the combined droplet, at least (i) the first reagent and (ii) the second reagent are used to form at least a portion of the biopolymer,

Wherein producing at least the portion of the biopolymer occurs in 10 minutes or less.

2. The method of claim 1, wherein the biopolymer is a polynucleotide.

3. The method of claim 1, wherein the biopolymer is a polypeptide.

4. The method of any one of claims 1 or 2, wherein the polynucleotide comprises about 10 to about 250 bases.

5. The method of any one of claims 1 to 3, wherein the polynucleotide comprises about 260 to about 1kb.

6. The method of any one of claims 1 to 3, wherein the polynucleotide comprises about 1kb to about 10,000kb.

7. The method of any one of claims 1 to 6, wherein vibration is applied to the synthetic droplets during (b), (c), or both.

8. The method of any one of claims 1 to 7, wherein the method further comprises one or more washing steps comprising moving wash droplets to contact the combined droplets.

9. The method of claim 8, wherein vibration is applied to the one or more washing steps.

10. The method of any one of claims 1 to 9, wherein the surface is a dielectric.

11. The method of any one of claims 1 to 9, wherein the surface comprises a dielectric layer disposed over one or more electrodes.

12. The method of any one of claims 1 to 9, wherein the surface is a surface of a polymer film.

13. The method of any one of claims 1 to 9, wherein the surface comprises one or more oligonucleotides bound to the surface.

14. The method according to any one of claims 1 to 9, wherein the surface is a surface of a lubricating liquid layer.

15. The method of any one of claims 1 to 14, wherein the plurality of droplets comprises a third droplet comprising a third reagent.

16. The method of any one of claims 1-15, wherein the first reagent, the second reagent, the third reagent, or any combination thereof comprises one or more functionalized beads.

17. The method of claim 16, wherein the functionalized bead comprises one or more oligonucleotides immobilized thereto.

18. The method of any one of claims 1 to 17, wherein vibration is applied to the first droplet, the second droplet, the third droplet, a wash droplet, or a mixture thereof.

19. The method of any one of claims 1 to 18, wherein the first reagent, the second reagent, the third reagent, or any combination thereof comprises a polymerase.

20. The method of any one of claims 1 to 19, wherein the first agent, the second agent, the third agent, or any combination thereof comprises a biomonomer.

21. The method of claim 20, wherein the biomonomer is an amino acid.

22. The method of claim 20, wherein the biomonomer is a nucleic acid molecule.

23. The method of claim 22, wherein the nucleic acid molecule comprises adenine, cytosine, guanine, thymine, or uracil.

24. The method of any one of claims 1 to 23, wherein the first reagent, the second reagent, the third reagent, or any combination thereof comprises one or more functionalized discs.

25. The method of claim 24, wherein the functionalized disc comprises one or more oligonucleotides immobilized thereto.

26. The method of any one of claims 1 to 25, wherein the first agent, the second agent, the third agent, or any combination thereof comprises an enzyme that mediates synthesis or polymerization.

27. The method of claim 26, wherein the enzyme is selected from the group consisting of polynucleotide phosphorylase (PNPase), terminal deoxynucleotidyl transferase (TdT), DNA polymerase β, DNA polymerase λ, DNA polymerase μ, and other enzymes from the DNA polymerase X family.

28. The method of any one of claims 1 to 27, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 20 minutes or less.

29. The method of any one of claims 1 to 28, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 15 minutes or less.

30. The method of any one of claims 1-29, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 10 minutes or less.

31. The method of any one of claims 1-30, wherein at least one nucleic acid molecule of the polynucleotide is generated within the pooled droplets in 1 minute or less.

32. The method of any one of claims 1 to 31, wherein the combined droplets are temperature controlled.

33. The method of any one of claims 1 to 32, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a magnetic field.

34. The method of any one of claims 1 to 33, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to illumination.

35. The method of any one of claims 1 to 34, wherein the first droplet, the second droplet, the third droplet, or the combined droplet is subjected to a pH change.

36. The method of any one of claims 1 to 35, wherein the first droplet, the second droplet, the third droplet, or the combined droplet comprises deoxynucleoside triphosphates (dntps).

37. The method of claim 36, wherein the deoxynucleoside triphosphate may have a protecting group.

38. The method of claim 37, wherein the protecting group can be removed in a reaction.

39. The method of any one of claims 1 to 38, wherein the first, second, third or combined droplet is in contact with a surface on only one side.

40. The method of any one of claims 1 to 39, wherein the volume of the first, second, third, or combined droplets is between 1 nanoliter (1 nl) and 500 microliters (500 μl).

41. The method of any one of claims 1 to 40, wherein the volume of the first, second, third, or combined droplets is between 1 microliter (1 μl) and 500 microliters (500 μl).

42. The method of any one of claims 1 to 41, wherein the volume of the first, second, third, or combined droplets is between 1 microliter (1 μl) and 200 microliters (200 μl).

43. The method of any one of claims 1 to 42, wherein the method further comprises attaching the biopolymer to a second biopolymer.

44. The method of claim 43, wherein the second biopolymer is produced using the method of any one of claims 1-43.

45. A method for processing a sample, the method comprising:

a. providing an array, the array comprising:

i. a plurality of electrodes, and

a surface configured to support the sample,

b. introducing the droplets to the surface; and

46. The method of any one of the preceding claims, wherein the sample is a droplet.

47. The method of any one of the preceding claims, wherein the droplets comprise about 1 nanoliter to 1 milliliter.

48. The method of any one of the preceding claims, wherein the droplet comprises a biological material.

49. The method of any one of the preceding claims, wherein the biological sample comprises one or more biomolecules.

50. The method of any one of the preceding claims, wherein the biomolecule comprises a nucleic acid molecule, a protein, a polypeptide, or any combination thereof. The method of any one of the preceding claims, wherein the droplets comprise about 1 nanoliter to 1 milliliter.

51. The method of any of the preceding claims, further comprising instructing at least a subset of the plurality of electrodes to provide an electric field to alter the wetting characteristics of the surface.

52. A method according to any preceding claim, wherein the electromechanical actuator comprises a cantilever.

53. The method of any preceding claim, wherein the electromechanical actuator comprises one or more coupling members coupled to the array.

54. The method of any of the preceding claims, wherein the one or more coupling members comprise an electromagnetic actuator, a piezoelectric actuator, an ultrasonic sensor, a rotating eccentric mass, one or more motors with a rocking linkage mechanism, or any combination thereof.

55. The method of any of the preceding claims, wherein the one or more motors are brush motors, brushless motors, stepper motors, or any combination thereof.

56. A method according to any preceding claim, wherein the electromagnetic actuator comprises an electromagnetic voice coil actuator.

57. The method of any one of the preceding claims, wherein the vibration frequency comprises a gradient.

58. The method of any of the preceding claims, wherein the gradient rises from near where the cantilever couples to the array.

59. The method of any of the preceding claims, wherein the vibration comprises a mode.

60. A method according to any preceding claim, wherein the pattern is sinusoidal.

61. A method according to any preceding claim, wherein the pattern is square.

62. The method of any of the preceding claims, wherein the surface is a top surface of a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

63. The method of any of the preceding claims, wherein the surface comprises a layer disposed over a dielectric, wherein the dielectric is disposed over the plurality of electrodes.

64. The method of any of the preceding claims, wherein the layer comprises a liquid.

65. The method of any of the preceding claims, wherein the layer comprises a coating.

66. The method of any one of the preceding claims, wherein the coating is hydrophobic.

67. The method of any of the preceding claims, wherein the layer comprises a film.

68. The method of any preceding claim, wherein the film is a dielectric film.

69. The method of any of the preceding claims, wherein the dielectric film comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

70. The method of any one of the preceding claims, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

71. The method of any of the preceding claims, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ether (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

72. The method of any of the preceding claims, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

73. The method of any of the preceding claims, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

74. The method of any of the preceding claims, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

75. The method of any of the preceding claims, wherein the liquid further comprises a surfactant, electrolyte, rheology modifier, wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

76. The method of any one of the preceding claims, wherein the first plurality of electrodes, the dielectric, the surface configured to support the droplet containing the sample, or any combination thereof, is removable from the array.

77. A method according to any preceding claim, wherein the frequency of the vibration displaces the surface or a portion of the surface by 0.05 millimeters (mm) to 10mm.

78. The method of any one of the preceding claims, wherein the frequency of the vibration is 1 hertz (hz) to 20 kilohertz (khz).

79. A method for moving a droplet over an array, wherein the array comprises a plurality of electrodes, wherein none of the plurality of electrodes are permanently grounded; and a surface configured to support the droplet containing the sample, the method comprising:

Wherein the array does not include a permanent reference electrode.

80. A method according to any one of the preceding claims, wherein the time-varying voltage generates a current return path adjacent the droplet and one or more inactive electrodes, thereby causing movement of the droplet.

81. The method of any one of the preceding claims, wherein the plurality of electrodes are coplanar.

82. A method according to any one of the preceding claims, wherein the time-varying voltage is bipolar.

83. The method of any of the preceding claims, wherein the time-varying voltage is from about 1Hz to about 20kHz.

84. The method of any of the preceding claims, wherein upon activating at least the subset of the plurality of electrodes, the system further comprises a current return path adjacent to the droplet and one or more inactive electrodes.

85. The method of any of the preceding claims, wherein the activation of at least the subset of the plurality of electrodes generates an antagonistic current drive scheme in one or more adjacent electrodes.

86. A system for processing a sample, comprising:

a. A plurality of electrodes;

b. a dielectric layer disposed over the plurality of electrodes, wherein the dielectric layer comprises a surface configured to support a droplet comprising the sample; and

87. The system of any one of the preceding claims, wherein the liquid creates an adhesion between the plurality of electrodes and the dielectric layer.

88. The system of any of the preceding claims, wherein the liquid comprises a dielectric material.

89. The system of any one of the preceding claims, wherein the liquid prevents or reduces electrical conductivity of air disposed in the gap.

90. The system of any of the preceding claims, wherein the dielectric layer comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof.

91. The system of any one of the preceding claims, wherein the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chitin, or any combination thereof.

92. The system of any of the preceding claims, wherein the synthetic polymeric material comprises polyethylene, polypropylene, polystyrene, polyetheretherketone (PEEK), polyimide, polyacetal, polysiloxane, polyphenylene oxide, polyphenylene sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamide, polyoxymethylene, polycarbonate, polymethylpentene, polyphenylene oxide (polyphenylene oxide), polyphthalamide (PPA), polylactic acid, synthetic cellulose ether (e.g., methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethylcellulose), paraffin, microcrystalline wax, epoxy resin, or any combination thereof.

93. The system of any of the preceding claims, wherein the fluorinated material comprises Polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylene propylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxy tetrafluoroethylene copolymer (PFA), perfluoromethyl vinyl ether copolymer (MFA), ethylene chlorotrifluoroethylene copolymer (ECTFE), ethylene tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polytetrafluoroethylene (PCTFE), or any combination thereof.

94. The system of any of the preceding claims, wherein the surface modification comprises a siloxane, a silane, a fluoropolymer treatment, a parylene coating, any other suitable surface chemical modification process, a ceramic, a clay mineral, bentonite, kaolin, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

95. The system of any of the preceding claims, wherein the liquid comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

96. The system of any of the preceding claims, wherein the liquid further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

97. The system of any one of the preceding claims, wherein the surface comprises a liquid layer.

98. The system of any of the preceding claims, wherein the liquid layer comprises silicone oil, fluorinated oil, ionic liquid, mineral oil, ferrofluid, polyphenylene ether, vegetable oil, esters of saturated fatty acids and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefins, polyethylene glycol hydrocarbons, other non-hydrocarbon synthetic oils, or any combination thereof.

99. The system of any of the preceding claims, wherein the liquid layer further comprises a surfactant, an electrolyte, a rheology modifier, a wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.

100. The system of any of the preceding claims, wherein the dielectric layer is removable.

101. The system of any one of the preceding claims, wherein the attachment is sufficient to secure the liquid on the surface, and wherein the liquid is resistant to gravity.

102. The system of any one of the preceding claims, wherein the liquid is selected to preferentially wet the surface to facilitate movement of the droplets over the surface.

103. A method for circularizing a nucleic acid sample, the method comprising:

a. providing a droplet adjacent to an electrowetting array, wherein the droplet comprises the nucleic acid sample;

b. combining the droplets with one or more reagent droplets;

c. processing the droplets using the electrowetting array to circularize the nucleic acid sample;

d. separating the droplets from the one or more reagent droplets to generate circularized nucleic acid samples in the droplets.

104. The method of any one of the preceding claims, wherein the electrowetting array comprises a dielectric matrix.

105. The method of any one of the preceding claims, wherein the electrowetting array further comprises one or more reagent droplets.

106. The method of any one of the preceding claims, wherein the one or more reagent droplets comprise one or more reagents for circularizing the nucleic acid sample.

107. The method of any of the preceding claims, further comprising:

a. Combining the sample droplet with the one or more reagent droplets;

b. separating the sample droplet from the one or more reagent droplets; and

c. combining the one or more reagent droplets with a second droplet.

108. The method of any one of the preceding claims, wherein the droplet comprises one or more reagents for circularizing the nucleic acid sample.

109. The method of any one of the preceding claims, wherein (b) further comprises performing one or more droplet operations on the electrowetting array to process the droplets, wherein the one or more droplet operations comprise contacting the one or more reagent droplets with the droplets.

110. The method of any one of the preceding claims, wherein the electrowetting array comprises one or more electrodes located below a surface of the electrowetting array, and wherein the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate the one or more reagent droplets, the sample droplets, or both.

111. The method of any one of the preceding claims, wherein the one or more droplet operations comprise applying vibrations to the one or more reagent droplets, the sample droplet, or both.

112. The method of any one of the preceding claims, wherein the one or more droplet operations comprise applying vibration to the electrowetting array.

113. The method of any one of the preceding claims, further comprising performing a sequencing reaction on the nucleic acid sample using a single polymerase.

114. The method of any one of the preceding claims, further comprising generating a sequencing read that is at least 70 kilobases (kb) in length.

115. The method of any one of the preceding claims, further comprising generating a sequencing read of at least 80 kilobases (kb) in length.

116. The method of any one of the preceding claims, further comprising generating a sequencing read of about 200 kilobases (kb) in length.

117. The method of any one of the preceding claims, wherein sequencing data of at least 100 (Gb) is generated.

118. The method of any one of the preceding claims, wherein sequencing data of at least 500Gb is generated.

119. The method of claim 16, wherein sequencing data of at least 512Gb is generated.

120. The method of any of the preceding claims, wherein at least 10Gb of data is generated.

121. The method of any of the preceding claims, wherein at least 30Gb of data is generated.

122. The method of any one of the preceding claims, wherein the sequencing reaction comprises repeated cycle sequencing.

123. The method of any one of the preceding claims, wherein one or more sub-reads of the sequencing reads are generated.

124. The method of any one of the preceding claims, wherein a consensus sequence is generated from the sub-reads of a sequencing read.

125. The method of any one of the preceding claims, wherein the sequencing reads have an a260/a280 ratio of less than about 1.84.

126. The method of any one of the preceding claims, further comprising generating a circularized nucleic acid sample.

127. The method of any one of the preceding claims, wherein the circularized nucleic acid sample comprises a target sequence.

128. The method of any one of the preceding claims, wherein the circularized nucleic acid sample comprises a plurality of sequences comprising the target sequence.

129. The method of any one of the preceding claims, wherein at least 80% of the plurality of sequences comprise the target sequence.

130. The method of any one of the preceding claims, wherein the method further comprises, prior to (a), obtaining the nucleic acid sample from a biological sample on the electrowetting array.

131. A system for inducing droplet motion, comprising:

a. a surface configured to support the droplet, the droplet comprising at least one bead formed of a material configured to couple to a magnetic field;

b. an actuator coupled to a magnet, wherein the magnet is configured to provide the magnetic field, and wherein the actuator is configured to translate the magnetic field along a plane parallel to the surface; and

c. a controller operably coupled to the actuator, wherein the controller is configured to instruct the actuator to translate the magnetic field along the plane such that the droplet undergoes movement along the surface as the magnetic field translates along the plane.

132. The system of claim 130, wherein the actuator is a switch.

133. The system of claim 130, wherein the actuator comprises a motor coupled to the magnet, wherein the motor is configured to translate the magnet in a direction parallel to the surface.

134. The system of claim 130, further comprising electrodes configured to provide an electric field to the surface, wherein the electric field and the magnetic field are sufficient to cause the movement of the droplet.

135. The system of claim 130, wherein the actuator is configured to move the magnet to translate along at least two axes parallel to the plane.

136. The system of claim 130, wherein the magnet comprises a permanent magnet.

137. The system of claim 130, wherein the magnet comprises at least one electromagnet.

138. The system of claim 130, wherein the actuator comprises a pivot, and wherein the pivot is coupled to the surface.

139. The system of claim 130, wherein the surface comprises a dielectric disposed over one or more electrodes.

140. The system of claim 130, wherein the one or more magnets are disposed below the surface.

141. The system of claim 130, wherein the surface comprises a liquid layer.

142. The system of claim 130, wherein the liquid layer comprises a liquid having an affinity for the surface.

143. The system of claim 130, wherein the member comprises a pivot and wherein the pivot is coupled to the surface.