Selective isolation of an immobilized molecule

Abstract

Described herein are devices, systems, and methods for selectively isolating a molecule. The method includes forming a first complex having a first molecule and a particle, the first particle being disposed on a first working electrode, the first working electrode electrically coupled to a first counter electrode via a first solution; modulating an energy of the first working electrode relative to the first counter electrode to release the first complex from the first working electrode into the first solution.

Description

Attorney Docket Number: 69385-727601SELECTIVE ISOLATION OF AN IMMOBILIZED MOLECULECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to U.S. Provisional Application No. 63 / 688,077, filed August 28, 2024, which is incorporated herein by reference in its entirety.INTRODUCTION

[0002] Oligonucleotides are being researched for and used as therapeutics. However, synthesizing and isolating oligonucleotide sequences efficiently and effectively for testing or mass production is challenging. For example, many current techniques for synthesizing oligonucleotides require oligonucleotides of different sequences to be synthesized separately. While separately synthesized oligonucleotides can be easily isolated from one another for downstream applications, such isolated synthesis techniques may be resource and cost ineffective.SUMMARY

[0003] Some embodiments of the technology disclosed herein relate to a method for selectively isolating a molecule. The method includes forming a first complex having a first molecule and a particle, the first particle being disposed on a first working electrode, the first working electrode electrically coupled to a first counter electrode via a first solution; modulating an energy of the first working electrode relative to the first counter electrode to release the first complex from the first working electrode into the first solution.

[0004] In an aspect, provided herein is a method for selectively isolating a molecule, the method comprising: (a) forming a complex comprising (i) the molecule and (ii) a particle, wherein the particle is disposed on a working electrode, and wherein the working electrode is electrically coupled to a counter electrode via a solution; (b) modulating an energy of the working electrode relative to the counter electrode to release the particle from the working electrode into the solution; and (c) cleaving the molecule from the particle.

[0005] In some cases, the modulating the energy of the working electrode comprises applying a potential to the working electrode relative to the counter electrode. In some cases, the applying the potential to the working electrode relative to the counter electrode comprises applying one or more pulses to the working electrode. In some cases, a pulse of the one or more pulses comprises application of a positive potential and a negative potential in any order to the working electrode. In some cases, the negative potential is from about -100 V to about -0.1 V. InAtorney Docket Number: 69385-727601 some cases, the negative potential is from about -10 V to about -3 V. In some cases, the positive potential is from about 0.01 V to about 100 V. In some cases, the positive potential is from about3 V to about 10 V. In some cases, a pulse of the one or more pulses has a pulse length from about 0.1 seconds to about 60 seconds. In some cases, each pulse of the one or more pulses has a pulse length from about 0.1 seconds to about 60 seconds. In some cases, the modulating the energy in (b) comprises a total applied energy duration from about 0.2 seconds to about 600 seconds.

[0006] In some cases, the working electrode is coupled to a surface of a fluidic device, and wherein the surface of the fluidic device further comprises one or more additional working electrodes. In some cases, the modulating the energy in (b) results in an electrochemical reaction on the working electrode, and wherein the modulating the energy in (b) does not result in an electrochemical reaction on one or more of the additional electrodes.

[0007] In some cases, the particle comprises silica, a polymer, or a metal oxide, or any combination thereof. In some cases, the working electrode is disposed on a solid substrate. In some cases, the working electrode is formed by a coating on a solid substrate. In some cases, the solid substrate comprises an inert, nonconductive solid.

[0008] In some cases, the solution comprises an electrolyte. In some cases, the solution has an ionic conductivity of 0.0001 S / m to 100 S / m. In some cases, the solution comprises (i) water, an organic solvent, or both and (ii) an inorganic salt, an organic salt, or both.

[0009] In some cases, the particle has a diameter from about 0.001 pm to about 100 pm. In some cases, the particle has a diameter from about 1 pm to about 50 pm. In some cases, the particle has a diameter from about 5 pm to about 15 pm. In some cases, (b) occurs in a fluidic device, and wherein the fluidic device further comprises one or more additional particles. In some cases, an average diameter of the particle and the one or more additional particles is from about 0.001 pm to about 100 pm. In some cases, the average diameter of the particle and the one or more additional particles is from about 1 pm to about 50 pm. In some cases, the average diameter of the particle and the one or more additional particles is from about 5 pm to about 15 pm. In some cases, the particle is porous. In some cases, the particle has an average pore diameter from about 1 A to about 2000 A. In some cases, the particle has an average pore diameter from about 500 A to about 1500 A. In some cases, the particle and the one or more additional particles are porous, and an average pore diameter of the particle and the one or more additional particles is from about 1 A to about 2000 A. In some cases, the average pore diameter of the particle and the one or more additional particles is from about 500 A to about 1500 A.Atorney Docket Number: 69385-727601

[0010] In some cases, the working electrode is individually addressable from one or more of the additional working electrodes. In some cases, each working electrode of the additional working electrodes is individually addressable. In some cases, the method further comprises (d) forming an additional complex comprising (i) an additional molecule and an additional particle, wherein the additional particle is disposed on an additional working electrode of the additional working electrodes, and wherein the additional working electrode is electrically coupled to an additional counter electrode. In some cases, the method further comprises (e) modulating an energy of the additional working electrode relative to the additional counter electrode to release the additional complex from the additional working electrode into the fluidic device. In some cases, the additional working electrode and the additional counter electrode are electrically coupled via the solution. In some cases, the additional working electrode and the additional counter electrode are electrically coupled via additional solution. In some cases, the first molecule is different from the additional molecule.

[0011] In some cases, the first complex and the additional complex comprise a part of a plurality of molecule particle complexes, and the method further comprises sequentially selectively releasing at least a portion of the plurality of molecule particle complexes. In some cases, the plurality of molecule particle complexes comprise at least 50 molecule particle complexes. In some cases, the at least 50 molecule particle complexes are released within a time period of no more than 30 minutes. In some cases, the plurality of molecule particle complexes comprise at least 90 molecule particle complexes. In some cases, the at least 90 molecule particle complexes are released within a time period of no more than 30 minutes.

[0012] In some cases, prior to (a), the particle is disposed on the working electrode by heat treatment, and the heat treatment does not exceed 350 °C. In some cases, prior to (a), the particle is coupled to the working electrode by heat treatment, and the heat treatment does not exceed 300 °C.

[0013] In some cases, the molecule comprises a polynucleotide strand. In some cases, the molecule comprises a deoxyribonucleic acid (DNA) strand. In some cases, the molecule comprises a ribonucleic acid (RNA) strand. In some cases, the particle comprises a silica microparticle.

[0014] In some cases, the method further comprises collecting the released complex in a container. In some cases, the method further comprises collecting the released complex from (b) and the released additional complex from (e) in separate containers.Atorney Docket Number: 69385-727601

[0015] In some such embodiments, modulating the energy includes applying a potential to the first working electrode relative to the first counter electrode. In some cases, applying a potential to the first working electrode relative to the first counter electrode includes applying one or more pulses to the first working electrode. Each pulse may include the application of a positive potential and a negative potential in any order to the first working electrode. In some cases, the negative potential is between -100 V to -0.1 V. In some cases, the negative potential is from -10 V to -3 V. In some cases, the positive potential is between 0.01 V to 100 V. In some cases, the positive potential is from 3 V to 10 V. In some cases, each pulse has a pulse length from 0.1 seconds to 60 seconds. In some cases, modulating the energy includes applying energy with a total applied energy duration of 0.2 seconds to 600 seconds. In some cases, modulating the energy results in an electrochemical reaction on the surface of the first electrode only.

[0016] In some cases, the first particle includes silica, a polymer, a metal oxide, or any combination thereof. In some cases, the first electrode is disposed on a solid substrate. In some cases, the first electrode is formed by a coating on a solid substrate. In some cases, the solid substrate includes an inert, nonconductive solid. In some cases, the first solution is an electrolyte. In some cases, the first solution has an ionic conductivity of 0.0001 S / m to 100 S / m. In some cases, the first solution includes water, an organic solvent, or both and includes an inorganic salt, an organic salt, or both. In some cases, the first particle is a part of a population of particles and the population of particles has an average diameter of 0.001 pm to 100 pm. In some cases, the first particle is a part of a population of particles and the first particle is porous. In some cases, the population of particles has an average pore size of 1 A to 2000 A. In some cases, the first working electrode is a part of a working electrode array comprising a second working electrode and a third working electrode.

[0017] In some cases, the method further includes forming a second complex comprising a second molecule and a second particle, the second particle being disposed on a second working electrode, the second working electrode being electrically coupled to a second counter electrode through a second solution. In some cases, the method further includes forming a third complex comprising a third molecule and a third particle, the third particle being disposed on a third working electrode, the third working electrode being electrically coupled to a third counter electrode through a third solution. In some cases, the first molecule is different from the second molecule and different from the third molecule. In some cases, the method further includes selectively releasing the second complex by modulating an energy of the second electrode relative to the second counter electrode to release the second complex from the second electrode into the second solution or selectively releasing the third complex by modulating an energy ofAtorney Docket Number: 69385-727601 the third electrode relative to the third counter electrode to release the third complex from the third electrode into the third solution. In some cases, the first complex, the second complex, and the third complex include a part of a plurality of molecule particle complexes. In some cases, the method further includes sequentially selectively releasing at least a some of the plurality of molecule particle complexes.BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is flow diagram illustrating an overview of a method for selectively isolating a molecule consistent with embodiments of the present disclosure.

[0019] FIG. 2 is a cross-sectional side view of a first fluidic device that may be used to accomplish the methods of one or more embodiments of the present disclosure.

[0020] FIG. 3 is a schematic drawing illustrating a first workflow for isolating a molecule that is consistent with embodiments of the present disclosure.

[0021] FIG. 4 is a schematic drawing illustrating a second workflow for isolating a molecule that is consistent with embodiments of the present disclosure.

[0022] FIG. 5 is a schematic top view of the functional elements of a second fluidic device that may be used to accomplish the methods of one or more embodiments of the present disclosure.

[0023] FIG. 6 is a graphical representation of the results of Example 1, showing total ion chromatograms for each sample.

[0024] FIG .7 is a graphical representation of the results of Example 2, showing total ion chromatograms for each sample.

[0025] FIG. 8 is a graphical representation of the results of Example 3, showing total ion chromatograms for each sample.

[0026] FIG. 9 is a schematic of a cross-section of an electrode functionalized with silica microparticles, in accordance with some embodiments.

[0027] FIG. 10 shows a computer system that is programmed or otherwise configured to implement methods provided herein, according to some embodiments.DEFINITIONS

[0028] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding ofAtorney Docket Number: 69385-727601 certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0029] The terms “fluidic device” and “fluidic system” are used here to refer to systems and devices used for handling and controlling the flow of fluids, for example into, through, around, across, via, (etc.) a region. Such fluidic devices and systems may be used to handle fluids as part of specific processes, such as those associated with, for example, molecular biology applications, chemical synthesis applications, and sensing applications. The term “fluidic” is understood to also encompass “microfluidic.” A microfluidic device or system may be used to handle fluid volumes and flow rates at a microliter scale (e.g., ranging from 1-10,000 microliters).

[0030] Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terms “a,” “an,” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of’ and “comprises at least one of’ followed by a list refers to any one of the items in the list and any combination of two or more items in the list.

[0031] Herein, the term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and embodiments. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0032] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. Any of the elements or combinations of elements that are recited in this disclosure in open-ended language (e.g., comprise and derivatives thereof), are considered to additionally be recited in closed-ended language (e.g., consist and derivatives thereof) and in partially closed-ended language (e.g., consist essentially, and derivatives thereof).

[0033] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of theAtorney Docket Number: 69385-727601 listed elements. The use of “and / or” in some instances does not imply that the use of “or” in other instances may not mean “and / or.”

[0034] The recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). Where a range of values is “up to” or “at least” a particular value, that value is included within the range.

[0035] As used here, “have,” “having,” “include,” “including,” “comprise,” “comprising,” or the like are used in their open-ended sense, and generally mean “including, but not limited to.” It will be understood that “consisting essentially of,” “consisting of,” and the like are subsumed in “comprising” and the like. As used herein, “consisting essentially of,” as it relates to a composition, product, method, or the like, means that the components of the composition, product, method, or the like are limited to the enumerated components and any other components that do not materially affect the basic and novel characteristic(s) of the composition, product, method, or the like.

[0036] “Optional” or “optionally” means that the subsequently described event, circumstance, or component, can or cannot occur, and that the description includes instances where the event, circumstance, or component, occurs and instances where it does not.

[0037] The words “preferred” and “preferably” refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the inventive technology.

[0038] While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a method comprising an incorporation step, a detection step, a deprotection step, and one or more wash steps includes embodiments where the method consists of enumerated steps and embodiments where the method consists essentially of the enumerated.

[0039] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.Atorney Docket Number: 69385-727601

[0040] Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “some embodiments,” “some cases,” or “one or more embodiments” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0041] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. However, it will be understood that a presented order is one embodiment of an order by which the method may carried out. Any recited single or multiple feature or aspect in any one claim may be combined or permuted with any other recited feature or aspect in any other claim or claims.DETAILED DESCRIPTION

[0042] The present disclosure relates to methods of selectively isolating molecules from other molecules. More specifically, the methods relate to the site selective release of molecules from a solid surface while other molecules remain immobilized on the surface. The methods of the present disclosure may be integrated, for example, into solid phase synthesis techniques and / or binding assays. The present disclosure also relates to devices and systems configured for performing such methods.

[0043] Conventional solid phase synthesis techniques typically involve cleaving the synthesized molecules from the solid phase in tandem resulting in a mixture of molecules. To separate groups of molecules of different chemical identities, further purification is performed prior to downstream applications.

[0044] In contrast to the cleaving step of conventional solid phase synthesis techniques, the methods of the present disclosure may allow for the selective cleavage of molecules from a solid phase. For example, an array of a number of groups of molecules having different chemical identities may be synthesized via solid phase synthesis on a solid support. Using the methods of the present disclosure, a first group of molecules, a second group of molecules, and a third and additional groups of molecules may each independently be released from the solid support andAtorney Docket Number: 69385-727601 isolated. For instance, the first group of molecules may be released and collected prior to the release and capture of the second group of molecules. Since the groups of molecules are independently released, mixtures of different groups of molecules can be avoided in the final product if desired. Additionally, the methods of the present disclosure may allow for the selective release of molecules without damaging the released molecules or the molecules that remain bound to the solid support.

[0045] FIGS. 1, 2, 3, and 4 are referenced to illustrate embodiments consistent with the present disclosure. For clarity, the description of each element and step in the figures is described in the singular. However, it should be understood that the method steps may be applied to a plurality of molecules and to arrays that include various pluralities of molecules.

[0046] FIG. 1 is a flow chart illustrating an overview of the molecule isolation method 100. In some embodiments, the method 100 is a selective molecule isolation method. The method 100 may include forming a first complex (step 200). The first complex may include a first molecule and a particle. The particle may be disposed on a first working electrode. The first working electrode may be electrically connected to a first counter electrode via a first electrically conductive medium, such as a first solution. The method may further include modulating an energy of the first working electrode to release the first complex from the first electrode into a first solution (step 300). The energy of the first working electrode may be modulated relative to the first counter electrode.

[0047] For purposes of illustration, aspects of the isolation method consistent with embodiments of the present disclosure are described with reference to FIG. 2. FIG. 2 is a cross- sectional top view of an illustrative fluidic device 1 that may be used to accomplish method 100. It is understood that any suitable configuration of a first working electrode, a first counter electrode, and a first complex disposed on the first working electrode may be used to accomplish isolation method 100. For example, in some embodiments, the isolation method of the present disclosure may be accomplished in the absence of a fluidic device. In some such embodiments, the first working electrode and the first counter electrode may be disposed in a container and electrically coupled via the electrically conductive medium (e.g., first solution) also within the container. Upon modulation of the energy of the working electrode relative to the counter electrode, the first complex disposed on the first working electrode can be freed into the first solution. The first solution can then be decanted from the container to isolate the first complex externally from the container.Atorney Docket Number: 69385-727601

[0048] As shown in FIG. 2, a fluidic device that may be used to accomplish at least a portion of the isolation method 100 may include a channel 5, a first working electrode 2, and a first counter electrode 9.

[0049] The channel 5 can be configured to contain the components of the method 100 during at least a portion of method 100. For example, the channel can be configured to contain the first solution, the first working electrode, the first counter electrode, and the first complex during at least step 300 of the isolation method 100 (see FIG. 1). Although a square shape of the channel is shown, the channel 5 is not particularly limited by its shape or size, or the arrangement of the elements within the channel. The channel may be formed using any suitable technique. For example, the channel may be formed as a groove or trench in a block of material. The channel may be a capillary in a glass capillary array (GCA). The channel may be a capillary tube in a bulk material in which one or more channels have been formed. In other embodiments, the channel may be patterned in a material using lithographic processing. For example, the channel may be formed in silicon using an anodic porous silicon patterning process. In other embodiments, the channel may be patterned into or onto a plastic surface for example. A channel may be formed by, molding, stamping, etching or other patterning processes such as two-photon polymerization.

[0050] The channel 5 can have an interior surface 4. The interior surface 4 may be made of any suitable material. In some embodiments, the interior surface is least partially formed in or from a solid substrate. In some embodiments, the solid substrate is an insulating solid substrate. In some embodiments, the interior surface is at least partially formed in or from an inert, nonconductive solid substrate. In some embodiments, the solid substrate includes silicon, silicon oxide, silicon, carbide, silicon nitride, metal oxides, metal carbides, metal nitrides, glasses (such as soda lime glass, borosilicate glass, controlled pore glass), or any combination thereof. In some embodiments, the substrate includes an insulating polymer, such as polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and polydimethylsiloxane (PDMS), styrene- (ethylene-butylene)-styrene (SEBS) polymers, crosslinked versions of polytetrafluoroethylene (PTFE), polyetherimide (PEI), polyetheretherketone (PEEK), polyphenylene sulfide (PPS).

[0051] A first working electrode 2 and a first counter electrode 9 can be contained within the channel 5. The first working electrode 2 and the first counter electrode 9 may not be in electrical contact with each other (i.e., the first counter electrode is not in direct physical contact with the first working electrode.) An electrically conductive medium, such as an electrically conductive solution (e.g., the first solution 3), can be provided within the channel 5 that electrically connects the first working electrode 2 and the first counter electrode 9. The first working electrode and theAtorney Docket Number: 69385-727601 first counter electrode can be electrically coupled to power sources. The first working electrode 2 and the first counter electrode 9 may be arranged in any configuration such that the electrodes are not in direct physical contact yet are in electrical contact. For example, as shown in FIG. 2, the first working electrode 2 and the first counter electrode 9 may be arranged on opposite faces of the channel 5. In other embodiments, the first working electrode and the first counter electrode may be co-planar. The electrodes can be in physical contact with an interior surface of the channel. For example, the electrodes may be disposed on the interior surface 4 of the channel.

[0052] The first working electrode 2 and the first counter electrode 9 may be made of the same or different materials. In some embodiments, the first working electrode and the first counter electrode may independently both include one or more noble metals. Illustrative noble metals can include, for example, platinum (Pt), gold (Au), iridium (Ir), rhodium (Rh), and ruthenium (Ru). Additionally, the first working electrode and the first counter electrode may independently include a conductive oxide of a noble metal, or an alloy of a noble metal. Additionally, the first working electrode and the first counter electrode may independently include one or more noble metals, one or more oxides of a noble metal, one or more alloys of a noble metal, or any combination thereof. Additionally, the first working electrode and the first counter electrode may independently include a conductive oxide of a metal, or an alloy of metals, or metal oxides which form a conductive film. In some embodiments, one or both of the first working electrode and the first counter electrode may independently include an oxide such as SiCh, AI2O3, TiCh or similar, doped with a metal such as platinum, nickel, tungsten, or similar metals. In some embodiments, the first working electrode and / or the first counter electrode may include or consist of platinum (Pt), for example.

[0053] In some embodiments, the first working electrode 2 is disposed on a solid substrate. The solid substrate may have any chemical composition as a solid substrate described herein. For example, the solid substrate may include any composition of an interior channel surface described herein. In some embodiments, the first working electrode may be a coating on a portion of the interior surface 4 of the channel. In some embodiments, the working electrode may be a coating having a thickness of 0.002 micrometers or greater, 0.01 micrometers or greater, 0.1 micrometers or greater, or 0.5 micrometers or greater. In some embodiments, the working electrode may be a coating having a thickness of 500 micrometer or less, 400 micrometers or less, 300 micrometers or less, 200 micrometers or less, 100 micrometers or less, 50 micrometers or less, 40 micrometers or less, 20 micrometers or less, 10 micrometers or less, 1 micrometers or less, 0.5 micrometers or less, 0.2 micrometers or less, or 0.1 micrometers or less.Atorney Docket Number: 69385-727601In some embodiments, the working electrode is formed by a coating having a thickness in a range of 0.002 micrometers to 1 micrometer, 0.01 micrometers to 0.5 micrometers, or 0.1 micrometers to 0.5 micrometers.

[0054] In some embodiments, the first counter electrode 9 is disposed on a solid substrate. The solid substrate may have any chemical composition as a solid substrate described herein. For example, the solid substrate may include any composition of an interior channel surface described herein. In some embodiments, the first counter electrode 9 may be a coating on a portion of the interior surface 4 of the channel. In some embodiments, first counter electrode 9 may be a coating having a thickness of 0.002 micrometers or greater, 0.01 micrometers or greater, 0.1 micrometers or greater, or 0.5 micrometers or greater. In some embodiments, first counter electrode 9 may be a coating having a thickness of 500 micrometer or less, 400 micrometers or less, 300 micrometers or less, 200 micrometers or less, 100 micrometers or less, 50 micrometers or less, 40 micrometers or less, 20 micrometers or less, 10 micrometers or less, 1 micrometers or less, 0.5 micrometers or less, 0.2 micrometers or less, or 0.1 micrometers or less. In some embodiments, the first counter electrode 9 is formed by a coating having a thickness in a range of 0.002 micrometers to 1 micrometer, 0.01 micrometers to 0.5 micrometers, or 0.1 micrometers to 0.5 micrometers.

[0055] Optionally, in some embodiments where the working electrode is a coating on an interior surface of the channel, an adhesion layer may be present between the inner surface of the channel and the electrode layer. The adhesion layer can function to increase adhesion of the electrode layer to the material of the inner surface itself. In some embodiments, the adhesion layer may include Ti, TiN, TiO?, Ta, Ta2Ns, or AI2O3, or the like, or a combination thereof. The specific material chosen may depend at least in part on the material of the interior surface of the channel, the material of the electrode, or both.

[0056] One or more passages may fluidically couple the channel to an external fluid source and / or an external container or reservoir. The one or more passages may be configured to allow fluid to enter and / or exit the channel. For example, as shown in FIG. 2, an inlet 6 and an outlet 7 are fluidically coupled to the channel. The inlet 6 can be configured to allow fluid to enter the channel and the outlet can be configured to allow fluid to exit the channel. Fluid may flow from the inlet to the outlet through the channel. Fluid flow may be controlled by using a pump or other suitable mechanism.

[0057] In some cases, the isolation method 100 includes providing a first complex 10 disposed on the first working electrode 2 (step 200 of FIG. 1). The first complex 10 can includeAtorney Docket Number: 69385-727601 a first molecule 12 and a particle 14 (FIG. 3). In some embodiments, the first complex includes a linker 16 that covalently couples the first molecule 12 to the particle 14.

[0058] The first molecule 12 can be a molecule of interest. A molecule of interest may be any molecule for which isolation is desired. Examples of molecules of interest may include biomolecules and synthetic molecules. Biomolecules may be any molecule that is found in nature, including, for example, proteins, peptides, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, and polysaccharides), oligonucleotides (i.e., RNA or DNA), lipids, fatty acids, sterols, steroids, other various small molecules, metabolites thereof, and endogenously modified variants thereof. Biomolecules may be endogenously produced, recombinantly produced, or synthetically produced. That is, biomolecules can include synthetically produced molecules that are identical to their naturally occurring counterparts. Synthetic molecules may be any molecule not found in nature. Synthetic molecules can include, for example, engineered peptides, engineered proteins, and engineered oligonucleotides that have a sequence not found in nature and / or one or more chemical modifications not found in nature and small molecules not found in nature. In some embodiments the first molecule is a naturally occurring oligonucleotide or a synthetic oligonucleotide.

[0059] In some cases, the first complex 10 includes a particle 14. The particle 14 may be made of or include any suitable material. Examples of suitable materials include metals, metal oxides, and polymers. In some embodiments, the particle includes silicon. In some embodiments, the particle includes silicon oxide, otherwise termed silica. In some embodiments, the particle includes fumed silica, sintered silica, or both. In some embodiments, the particle includes crosslinked polystyrene. In some embodiments, the particle includes nickel oxide, titanium oxide, or both.

[0060] The particle may be a part of a population of particles. The population of particles can have a number average diameter as determined by SEM and confirmed by sedimentation methods, such as, for example, laser diffraction and sieving analyses. The population of particles may have an average diameter of 0.001 micrometers to 500 micrometers. In some embodiments, the population of particles may have an average size of 0.001 micrometers or greater, 0.01 micrometers or greater, 0.1 micrometers or greater, 1 micrometer or greater, 10 micrometers or greater, 20 micrometers or greater, 40 micrometers or greater, 60 micrometers or greater, 80 micrometers or greater, 100 micrometers or greater, 200 micrometers or greater, 300 micrometers or greater, 400 micrometers or greater, or 500 micrometers or greater. In some embodiments, the population of particles may have an average diameter of 500 micrometer or less, 400 micrometer or less, 300 micrometer or less, 200 micrometer or less, 100 micrometer orAtorney Docket Number: 69385-727601 less, 80 micrometer or less, 60 micrometers or less, 40 micrometers or less, 20 micrometers or less, 10 micrometers or less, 1 micrometer or less, 0.1 micrometers or less, 0.01 micrometers or less, or 0.001 micrometers or less. In some embodiments, the population of particles may have an average diameter in a range of 0.001 micrometers to 500 micrometer, 0.01 micrometers to 400 micrometers, 0.1 micrometers to 300 micrometers, 1 micrometer to 200 micrometers, 10 micrometers to 100 micrometers, 20 micrometers to 80 micrometers, or 40 micrometers to 60 micrometers. In some embodiments, the population of particles may have an average diameter in a range of 1 micrometer to 50 micrometers.

[0061] The particle may be porous or nonporous. In embodiments where the particle is porous, the population of particles has an average pore diameter as determined by porosimetry. The population of particles may have an average pore diameter of 1 Angstrom to 2000 Angstroms. In some embodiments, the population of particles may have an average pore diameter of 1 Angstrom or greater, 10 Angstroms or greater, 100 Angstroms or greater, 250 Angstroms or greater, 500 Angstroms or greater, 750 Angstroms or greater, 1000 Angstroms or greater, 1250 Angstroms or greater, 1500 Angstroms or greater, 1750 Angstroms or greater, or 2000 Angstroms or greater. In some embodiments, the population of particles may have an average pore diameter of 2000 Angstroms or less, 1750 Angstroms or less, 1500 Angstroms or less, 1250 Angstroms or less, 1000 Angstroms or less, 750 Angstroms or less, 500 Angstroms or less, 250 Angstroms or less, 100 Angstroms or less, 10 Angstroms or less, or 1 Angstrom or less. In some embodiments, the population of particles may have an average pore diameter in a range of 1 Angstrom to 2000 Angstroms, 10 Angstroms to 1500 Angstroms, 100 Angstroms to 1000 Angstroms, or 250 Angstroms to 750 Angstroms. In some embodiments, the population of particles may have an average pore diameter in a range of 200 Angstrom to 2000 Angstroms, 220 Angstrom to 1800 Angstroms, 250 Angstrom to 1500 Angstroms, 300 Angstrom to 1200 Angstroms, 350 Angstrom to 1000 Angstroms, or 400 Angstrom to 800 Angstroms.

[0062] In some embodiments, the first molecule is not covalently coupled to the particle. In such embodiments, the first molecule may be non-covalently bound to the particle or another molecule displayed on the particle. For example, in some embodiments, the particle may be coupled to a probe configured to bind to the first molecule, thereby noncovalently coupling the first molecule to the particle.

[0063] In some embodiments, the first molecule is covalently coupled to the particle. The first molecule may be directly covalently attached to the particle such that there is no intervening linker between the first molecule and the particle. For example, the first molecule and theAtorney Docket Number: 69385-727601 particle may each include a reactive handle such that the reactive handles form a reaction product that covalently couples the first molecule to the particle.

[0064] In some embodiments, the first molecule may be covalently coupled to the particle through a linker 16 (FIG. 2). The linker may be of any length and any chemical composition. Linkers may be described by the identity of the linker precursor used to couple the particle and the first molecule. A linker precursor can include at least one reactive handle that reacts with a reactive handle on the particle and at least one reactive handle that reacts with a reactive handle on the first molecule thereby linking the particle and the first molecule. It is appreciated that the linker can include the reaction products of the reactive handles. Examples of reactive handles are known and can include alkoxy silanes, carboxylic acids or activated carboxylic acids, esters, amines, alkynes, azide, epoxides, alcohols, and the like. In some embodiments, the linker may have two or more reactive handles that react with the particle and / or the first molecule. For example, alkoxysilane linker precursors may have two or more alkoxy reactive handles that react with two or more reactive handles on the particle.

[0065] In some embodiments, the particle has an organo-silane monolayer surface. In some embodiments, the linker precursor is any suitable silane that terminates in alcohol, amine, or a labile ester that can be deprotected to reveal an alcohol. In some such embodiments, the linker precursor is an alkoxy silane. Examples of alkoxy silanes that may be used as linker precursors include N-(3 -tri ethoxy silylpropyl)-4-hydroxybutyramide; 7V,r-Bi s(2-hydroxy ethyl )-3- aminopropyltriethoxysilane; 3 -aminopropyltri ethoxysilane; 7V-(2-aminoethyl)-3- aminopropyltrimethoxysilane; 7V-(2-aminoethyl)-2,2,4-trimethyl-l-aza-2-silacyclopentane; 3-(4- semicarbazidyl)propyltriethoxysilane; 3-glycidyloxypropyl)tri ethoxysilane; (3- glycidoxypropyl)tri ethoxy silane; 3-glycidyloxypropyl)trimethoxysilane; 3- isocyanatopropyltriethoxysilane; 2-[(acetoxy(polyethyleneoxy)propyl]triethoxysilane; acetoxymethyltri ethoxysilane; acetoxymethyltrimethoxysilane; 1,1 l-bis(trimethoxysilyl)-4-oxa- 8-azaundecan-6-ol; (l-(3-triethoxysilyl)propyl)-2,2-diethoxy-l-aza-2-silacyclopentane; bis(3- triethoxysilylpropyl)amine; and the like. In some embodiments, the linker precursor is (3- Aminopropyl)triethoxysilane (APTES). In some embodiments, the functional groups may be diluted by doping in silanes that are simply glycols, alkanes, or capped with glycols, carbonyls or alkanes.

[0066] Returning to FIG. 2, the first complex 10 can be disposed on the first working electrode 2. The particle 14 of the first complex 10 can be in contact with the first working electrode 2. In some embodiments, the particle 14 can be adhered to the first working electrode 2. The particle 14 may be adhered to the first working electrode by contacting the particle withAtorney Docket Number: 69385-727601 the first working electrode and exposing to heat, for example, approximately 500 degrees Celsius for ten hours. Alternatively, the particle 14 may be adhered to the first electrode by drop casting a mixture of the particle and sodium silicate (for example, 2 vol-% Na2O(SiO2)x• XH2O solution / 98% silica particle dispersion (100 mg / mL) onto the first working electrode and followed by exposure to heat, for example, approximately 250 degrees Celsius for one hour.

[0067] In some embodiments, the first complex 10 is formed before the particle 14 is in contact with the first working electrode 2. For example, the particle 14 and the first molecule 12 may be covalently coupled and then the entire complex 10 may be deposited on the first working electrode 14 such that the particle 14 is contacting the first working electrode 2.

[0068] In other embodiments, the first complex 10 is formed while the particle 14 is in contact with the first working electrode 2. For example, the particle 14 may be adhered to the first electrode 2 in the absence of the first molecule 12. Once adhered, the first molecule 12 can be covalently attached to the particle 14 to form the first complex 10. In some embodiments, it may be beneficial to install the particle 14 on the first working electrode 2 prior conjugating the first molecule 12 in order to prevent the adhering procedure from reacting with and / or degrading the first molecule 12.

[0069] The first complex 10 may be formed in a variety of ways. In some embodiments, the first molecule 12 may be covalently attached to the particle 14. In other embodiments, a first molecule precursor may be covalently attached to the particle 14 and then further chemically modified to form the first molecule 12. For example, a first molecule precursor may be covalently attached to the particle 14 and then reacted with one or more reagents to change the chemical structure thereby forming the first molecule 16. In yet other embodiments, the first molecule 12 may be synthesized while covalently attached to the particle 14. For example, oligonucleotides and peptides can be synthesized monomer by monomer (i.e., nucleotide by nucleotide or amino acid by amino acid) using known solid phase synthesis techniques, while covalently attached to the particle 14.

[0070] In some cases, the molecule is a polynucleotide with greater than 5, greater than 10, greater than 25, greater than 50, or greater than 100 nucleotides. In some cases, the molecule is a polynucleotide comprising 5 to 200 nucleotides. In some cases, the molecule is a polynucleotide comprising 5 to 100 nucleotides. In some cases, the molecule is a polynucleotide comprising 5 to 50 nucleotides. In some cases, the molecule is a polynucleotide comprising 5 to 25 nucleotides. In some cases, the molecule is a polynucleotide comprising 5 to 10 nucleotides.Atorney Docket Number: 69385-727601

[0071] Returning to FIG. 1, the method 100 can include modulating an energy of the first working electrode 2 relative to the first counter electrode 9 to release the first complex from the first working electrode 2 into the first solution (step 300). FIG. 3 schematically illustrates the transformation of step 300. Upon modulating the energy of the first electrode (step A), the first complex can be released from the first electrode, resulting in a freed first complex 10 (including the particle 14 and the first molecule 12) and a first electrode 2 that is not associated with the first complex 10. In some cases, modulation of the energy of the first working electrode 2 allows the first complex 10 to dissociate from the first working electrode 2. Although FIGS. 3 and 4 illustrate a single complex with a single particle and a single molecule of interest being attached to a working electrode, in reality, each electrode may have a plurality of complexes attached to it. Additionally, each particle may have a plurality of molecules attached to it. According to an embodiment, all of the complexes attached to a given electrode are of the same type (e.g., include the same molecule of interest). In some other embodiments, the complexes attached to a given electrode are of a different type (e.g. a mixture of different molecules of interest).

[0072] In some embodiments, modulating the energy of the first working electrode results in an electrochemical reaction on the surface of the first working electrode. Without wishing to be bound by theory, it is thought that the electrochemical reaction may decrease the adhesion between the first working electrode and the particle allowing the complex to become dislodged from the first working electrode. Alternatively, or in addition, modulating the energy of the first working electrode may result in the formation of gas cavities or bubbles that cause the complex to become dislodged from the first working electrode.

[0073] Modulating the energy of the first working electrode relative to the first counter electrode may include applying a potential to the first working electrode. The potential applied to the first working electrode may be a negative potential or a positive potential relative to the first counter electrode. In some cases, both a positive potential and a negative potential are applied. The intensity of the applied potential may be from 0.1 volts (V) to 100 V and the negative counterparts of such potential intensity (i.e., -100 V to -0.1 V). In some embodiments, the intensity of the applied potential may be 0.1 V or greater, 1 V or greater, 2 V or greater, 3 V or greater, 4 V or greater, 5 V or greater, 10 V or greater, 20 V or greater, 30 V or greater, 40 V or greater, 50 V or greater, 60 V or greater, 70 V or greater, 80 V or greater, 90 V or greater, or 100 V or greater. In some embodiments, the intensity of the applied potential may be 100 V or less, 90 V or less, 80 V or less, 70 V or less, 60 V or less, 50 V or less, 40 V or less, 30 V or less ,20 V or less, 10 V or less, 5 V or less, 4 V or less, 3 V or less, 2 V or less, or 1 V or less. InAtorney Docket Number: 69385-727601 some embodiments, the intensity of the applied potential may be 0.1 V to 100 V, 1 V to 80 V, 2 V to 70 V, 1 V to 3 V, 1 V to 60 V, 4 V to 50 V, 5 V to 40 V, 10 V to 30 V, or 3 V to 10 V.

[0074] In some embodiments, the potential is applied to the first working electrode continuously for a period of time. In some embodiments, modulating the energy of the first working electrode includes applying a single potential to the first working electrode. The applied potential may have any intensity as described herein. For example, the voltage may be applied continuously for 5 seconds to 10 minutes. In some embodiments, the voltage may be applied for 5 seconds or longer, 10 seconds or longer, 20 seconds or longer, 30 seconds or longer, 45 seconds or longer, 1 minute or longer, 2 minutes or longer, 3 minutes or longer, 4 minutes or longer, 5 minutes or longer, 6 minutes or longer, 7 minutes or longer, 8 minutes or longer, or 9 minutes or longer. In some embodiments, the voltage may be applied for 10 minutes or less, 9 minutes or less, 8 minutes or less, 7 minutes or less, 6 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less.

[0075] In some embodiments, modulating the energy of the first working electrode relative to the first counter electrode includes alternating the potential of the first working electrode relative to the first counter electrode between two or more intensities. In some embodiments, alternating the potential includes pulsing the first working electrode between a negative potential (negative voltage) and a positive potential (positive voltage) one or more times. Stated differently, in some embodiments, alternating the potential includes applying one or more pulses to the first working electrode. A pulse refers to the application of a potential of a first sign then applying a second potential of the opposite sign. A such, a pulse is made up of two half-pulses, each half pulse having a different potential sign. For example, a pulse is the application of a positive potential followed by the application of a negative potential to the same electrode.

[0076] The total number of pulses applied to the first working electrode may vary. In some embodiments, the total number of pulses may be 1 or more, 5 or more, 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, 250 or more, 500 or more, 750 or more, or 1000 or more. In some embodiments, the total number of pulses may be 1000 or less, 750 or less, 500 or less, 250 or less, 100 or less, 75 or less, 50 or less, 25 or less, 10 or less, 5 or less, or 1 or less. In some embodiments, the total number of pulses may be 1 to 500, 5 to 250, 10 to 100, 15 to 75, 20 to 50, 25 to 45, or 30 to 40.

[0077] A pulse has a pulse length. The pulse length is the total amount of time a potential is being applied to an electrode during a pulse. A pulse length is made of two half pulse lengths. A half pulse length is the amount of time of a half pulse. When multiple pulses are applied, eachAtorney Docket Number: 69385-727601 pulse may have the same pulse length or a different pulse length. Additionally, each half pulse length of a single pulse may be the same or different.

[0078] In some embodiments, each pulse independently has a pulse length of 0.1 seconds to 60 seconds. In some embodiments, each pulse independently has a pulse length of 0.1 seconds or greater, 1 second or greater, 5 seconds or greater, 10 seconds or greater, 20 seconds or greater, 30 seconds or greater, 40 seconds or greater, 50 seconds or greater, or 60 seconds or greater. In some embodiments, each pulse independently has a pulse length of 60 seconds or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, 1 second or less, or 0.1 seconds or less. In some embodiments, each pulse independently has a pulse length of 1 second to 50 seconds, 5 second to 40 seconds, or 10 second to 30 seconds.

[0079] In some embodiments, each half pulse independently has a pulse length of 0.05 seconds to 60 seconds. In some embodiments, each half pulse independently has a pulse length of 0.05 seconds or greater, 0.1 seconds or greater, 0.5 seconds or greater, 1 second or greater, 2.5 seconds or greater, 5 seconds or greater, 10 seconds or greater, 15 seconds or greater, 20 seconds or greater, 25 seconds or greater, or 30 seconds or greater. In some embodiments, each pulse independently has a pulse length of 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 2.5 seconds or less, 1 second or less, 0.5 seconds or less, 0.1 seconds or less, or 0.05 seconds or less. In some embodiments, each half pulse independently has a pulse length of 0.05 seconds to 30 seconds, 0.1 seconds to 25 seconds, 0.5 seconds to 20 seconds, 1 second to 15 seconds, or 2.5 seconds to 10 seconds.

[0080] The intensity of the potential for each half pulse of a pulse may be the same. For example, the first working electrode may be pulsed between -3 V and 3 V. In other embodiments, the intensity of the potential of each half pulse of a pulse may be different. For example, the first working electrode may be pulsed between -3 V and 1 V. Each half pulse of a pulse may independently have any potential intensity as described herein.

[0081] In some embodiments, modulating the energy of the first working electrode includes applying a single potential to the first working electrode. The applied potential may have any intensity as described herein.

[0082] Modulating the energy of the first electrode has a total applied pulsed energy duration. The total applied pulsed energy duration may be the total time a potential is applied to the first working electrode through applied pulses. For example, the total applied energy duration may be the sum of all the pulse lengths. In some embodiments, the applying pulses toAtorney Docket Number: 69385-727601 the first working electrode may result in the dislodgement of the first complex in a shorter time period than if a potential was applied continuously to the first working electrode.

[0083] In some embodiments, the total applied energy duration may be from 0.1 seconds to 600 seconds. In some embodiments, the total applied energy duration may be 0.1 seconds or greater, 0.2 seconds or greater, 0.5 seconds or greater, 1 second or greater, 5 seconds or greater, 10 seconds or greater, 25 seconds or greater, 50 seconds or greater, 75 seconds or greater, 100 seconds or greater, 200 seconds or greater, 300 seconds or greater, 400 seconds or greater, or 500 seconds or greater. In some embodiments, the total applied energy duration may be 500 seconds or less, 400 seconds or less, 300 seconds or less, 200 seconds or less, 100 seconds or less, 75 seconds or less, 50 seconds or less, 25 seconds or less, 10 seconds or less, 5 seconds or less, 1 second or less, 0.5 seconds or less, 0.2 seconds or less, or 0.1 seconds or less. In some embodiments, the total applied energy duration may be from 0.1 seconds to 600 seconds, 0.2 seconds to 500 seconds, 0.5 seconds to 400 seconds, 1 second to 300 seconds, 5 seconds to 200 seconds, 10 seconds to 100 seconds, or 25 seconds to 75 seconds.

[0084] Modulating the energy of the first electrode can release the first complex from the first working electrode into the first solution. In some caes, the first solution is configured to electrically couple the first working electrode and the first counter electrode. As such, the first solution may be or may include an electrolyte. The first solution may include water, an organic solvent, acetonitrile, N,N-dimethylformamidine (DMF), n-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide, acetone, ethyl acetate, methanol, ethanol, isopropanol, pyridine, nitromethane, ethylene glycol, or any one or more combination thereof. In some embodiments, the first solution includes one or more inorganic compounds, one or more organic compounds, or both. Illustrative inorganic compounds can include, for example, sodium hydroxide (NaOH), potassium hydroxide (KOH), similar compounds, and combinations thereof. In some embodiments, the first solution may include one or more organic compounds or salts thereof. Illustrative organic compounds can include, for example tetraethylammonium; 1 -butyl 3-methyl- imidazolium; l-butyl-3 -methyl imidazolium; 1,1-dimethyl-pyrrolidinium tris(pentafluoroethyl); 1,3-dimethyl-imidazolium; 3-methyl-A-butyl-pyridinium; tetraethyl ammonium; Af-hexyl- pyridinium; tetra-n-butylammonium; tetra-n-butylammonium; tetra-n-butylammonium; tetra-n- butylammonium; tetra-n-butylammonium; tetra-n-butylammonium; tetra-n-butylammonium; and salts thereof such as, for example tetrafluoroborate salts thereof, tosylate salts thereof, trifluorophoshate salts thereof, trifluoromethanesulfonate salts thereof, hexafluorophosphate salts thereof, hydroxide salts thereof, bromide salts thereof, iodide salts thereof, fluoride salts thereof, chloride salts thereof, or acetate salts thereof. Illustrative organic compound saltsAtorney Docket Number: 69385-727601 include, for example, tetraethylammonium / ?-toluenesulfonate; 1 -butyl 3-methyl-imidazolium tetrafluoroborate; 1 -butyl -3 -methyl imidazolium tosylate; 1,1-dimethyl-pyrrolidinium tris(pentafluoroethyl)trifluorophosphate; 1,3-dimethyl-imidazolium trifluoromethanesulfonate; 3 -methyl-Af-butyl -pyridinium hexafluorophosphate; tetraethyl ammonium tetrafluoroborate; N- hexyl-pyridinium hexafluorophosphate; tetra-n-butylammonium hexafluorophosphate; tetra-n- butylammonium acetate; tetra-n-butylammonium hydroxide; tetra-n-butylammonium bromide; tetra-n-butylammonium fluoride; tetra-n-butylammonium chloride; tetra-n-butylammonium iodide; and combinations thereof. In one embodiment, the solution includes KOH. Suitable concentrations of compounds range from 10 millimolar to a saturating amount.

[0085] In some embodiments, the solution has an ionic conductivity of 0.0001 to 100 siemens per meter (S / m). In some embodiments, the solution has an ionic conductivity of 0.0001 S / m or greater, 0.001 S / m or greater, 0.01 S / m or greater, 0.1 S / m or greater, 1 S / m or greater, 10 S / m or greater, or 80 S / m or greater. In some embodiments, the solution has an ionic conductivity of 100 S / m or less, 10 S / m or less, 1 S / m or less, 0.1 S / m or less, 0.01 S / m or less, or 0.001 S / m or less. In some embodiments, the solution has an ionic conductivity from 0.0001 S / m to 100 S / m, 0.001 S / m to 50 S / m, 0.01 S / m to 10 S / m, or 0.1 S / m to 1 S / m.

[0086] The isolation method 100 may further include removing at least a portion of the first solution, containing the freed first complex, from contact with the first working electrode and the first counter electrode. Removing at least a portion of the first solution may include flowing a portion of the first solution out of the channel of the fluidic device through an outlet. For example, upon completion of step 300 of isolation method 100 (FIG. 1), the first solution containing the first complex may be flowed out of the channel 5 through the outlet 7 (FIG. 2).

[0087] Once removed from contact with the first working electrode and the first counter electrode, the first complex may undergo further processing to release the first molecule from the particle and / or linker. Often, processes common for cleaving a molecule from a solid phase support (e.g., the particle) include conditions (e.g., radiation, heat, and / or harsh reagents) that are not compatible with electrodes and / or fluidic devices. Since the methods of the present disclosure allow isolation of the solid phase-molecule complex prior to cleaving the molecule from the solid phase, the methods of the present disclosure may be compatible with various fluidic devices. That is, because the first complex can be removed from contact with the electrodes and the channel, the cleaving may be done without affecting the electrodes or the channel. Additionally, since cleavage of the first molecule from the particle is not necessarily integrated into the fluidic device, the fluidic device may be reusable.Atorney Docket Number: 69385-727601

[0088] The isolation method 100, may be used to selectively isolate two or more complexes. For example, two or more complexes having a different molecule of interest may be isolated sequentially from one another. For example, the first working electrode may be a part of a working electrode array that includes two or more electrodes. Each complex can be disposed on a different working electrode within the array. Each working electrode can be electrically coupled to a counter electrode via an electrically conductive medium, e.g., a solution. By independently modulating the energy of each working electrode in the array relative to a counter electrode, individual complexes may be released from their respective electrodes in series or in parallel.

[0089] FIG. 1 and FIG. 4 illustrate how the isolation method 100 may be used to selectively isolate three complexes 10, 20, and 30. FIG. 1 outlines additional isolation method steps for selectively isolating three complexes, and FIG. 4 schematically illustrates the transformation of the additional method steps.

[0090] In some embodiments, the first working electrode 2a is a part of an electrode array that also includes a second working electrode 2b and a third working electrode 2c (FIG. 4). Although the embodiment shown and discussed here includes three working electrodes to isolate three complexes, any number of electrodes may be used. For example, the system may include 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 150 or more, or 200 or more electrodes. While there is no required limit, for practical purposes, the number of electrodes may be 5000 or fewer, 1000 or fewer, 750 or fewer, 500 or fewer, 200 or fewer, 100 or fewer, or 50 or fewer. As such, in some embodiments, the isolation method may further include forming a second complex 20 and a third complex 30 (steps 210 and 220 of FIG. 1) that are disposed on the second working electrode 2b and the third working electrode 2c, respectively. The second complex 20 can include a second molecule 22 and a second particle 24. In some embodiments, the second particle 24 and the second molecule 22 are covalently coupled by a linker 26. The second particle 24 of the second complex 20 can be disposed on a second working electrode 2b. The second working electrode 2b can be electrically connected to a counter electrode via an electrically conductive medium. The third complex 30 can include a third molecule 32 and a third particle 34. In some embodiments, the third molecule 32 and the third particle 34 are covalently coupled by a linker 36. The third particle 34 of the third complex 30 can be disposed on a third working electrode 2c. The third working electrode 2c can be electrically connected to a counter electrode via an electrically conductive medium. The second complex and the third complex may be formed using any method disclosed herein.Atorney Docket Number: 69385-727601

[0091] In some embodiments, the counter electrode for each of the first working electrode, the second working electrode, and the third working electrode is the same. In other embodiments, each working electrode is electrically connected to a separate counter electrode. For example, the first working electrode may be electrically connected to a first counter electrode, the second working electrode may be electrically connected to a second counter electrode, and the third working electrode may be electrically connected to a third counter electrode. Alternatively, the first working electrode, second working electrode, and third working electrode may all be electrically connected to one common counter electrode.

[0092] Each working electrode can be electrically coupled to a counter electrode via electrically conductive medium. In some embodiments, all working electrodes (e.g., the first working electrode, the second working electrode, and the third working electrode) may be electrically connected to a counter electrode through the same electrically conductive medium. For example, the working electrodes may be electrically connected to the same counter electrode through a single electrically conductive medium. In other embodiments, one or more working electrodes may be electrically connected to a counter electrode through electrically conductive media of different compositions or through different portions of an electrical media of a single composition. For example, the first working electrode may be electrically connected to a counter electrode (or the first counter electrode) through a first solution, the second working electrode may be electrically connected to a counter electrode (or the second counter electrode) through a second solution, and the third working electrode may be electrically connected to a counter electrode (or the third counter electrode) through a third solution. The first solution, the second solution, and the third solution may be physically isolated portions of an electrically conductive media. In some cases, the first solution, the second solution, and the third solution, may have different compositions.

[0093] The second molecule 22 and the third molecule 32 may be molecules of interest in addition to the first molecule 12. The second molecule 22 and the third molecule 32 may be any molecule of interest as described herein. In some embodiments, the first molecule 12 has a different chemical identity than the second molecule 22 and the third molecule 32. In some embodiments, the first molecule 12, the second molecule 22, and the third molecule 32 are oligonucleotides having different chemical identities.

[0094] The first particle 14, the second particle 24, and the third particle 34 may have the same composition or a different composition. The compositions of the particles may depend at least in part on the identity of the molecule attached to them. The composition of each particle may be any particle composition as described herein.Atorney Docket Number: 69385-727601

[0095] The first working electrode 2a, the second working electrode 2b, and the third working electrode 2c may have the same composition or a different composition. The composition of each working electrode may be any working electrode composition as described herein. The working electrodes can be electrically isolated from one another (i.e., the working electrodes are not in direct physical contact with each other). Each working electrode can be independently controllable. For example, a potential may be applied to the first working electrode but not the second or third working electrodes.

[0096] Any suitable configuration of a first working electrode, a second working electrode, a third working electrode, a first counter electrode, a second counter electrode, and a third counter electrode may be used to accomplish method 100. In some embodiments, all of the electrodes (working electrodes and counter electrodes) may be located within a single channel of a fluidic device. In other embodiments each working electrode is located within a different channel. In yet other embodiments, two working electrodes are located within one channel and one working electrode is located within a different channel. In any of the described configurations, the counter electrodes may be a single electrode that is electrically coupled to all of the working electrodes or multiple counter electrodes electrically coupled to one or more of the working electrodes. Each working electrode and counter electrode can be electrically coupled to a power source. In some embodiments, the working electrodes are coplanar. In some embodiments, the counter electrodes are coplanar. In some embodiments the working electrodes and the counter electrodes are not coplanar. For example, the counter electrodes may be disposed on or form an interior face of the channel that is opposite of the working electrodes.

[0097] After releasing the first complex 10 from the first working electrode 2a into the first working solution (step 300 in FIG. 1 and step B in FIG. 4), the second complex 20 and the third complex 30 may remain on the second working electrode 2b and the third working electrode 2c, respectively. At least a portion of the electrically conductive medium (e.g., first solution) containing the first complex may be removed from contact with the first working electrode, for example, by removing at least a portion of the electrically conducive medium from the fluidic device. In some embodiments where the first working electrode and the second working electrode are within the same channel of a fluidic device, another electrically conductive medium (e.g., second solution) or a fresh aliquot of the same electrically conductive medium (e.g., first solution) may be flowed into the channel to replace at least a portion of the electrically conductive medium that was removed from the channel after release of the first complex. In some embodiments where the first working electrode and the second electrode are not within theAtorney Docket Number: 69385-727601 same channel of the fluidic device, the electrically conductive medium may be independently present in the channel containing the second working electrode.

[0098] In some embodiments, the isolation method 100 may further include selectively releasing the second complex from the second working electrode or selectively releasing the third complex from the third electrode or both. The second complex can be released from the second working electrode 2b by modulating the energy of the second working electrode 2b relative to the second counter electrode to release the second complex into the electrically conductive medium (e.g., second solution; step 310 in FIG. 1 and steps C and F in FIG. 4). The third complex can be released from the third working electrode 2c by modulating the energy of the third working electrode 2c relative to the third counter electrode to release the third complex into the electrically conductive media (e.g., the third solution; step 310 in FIG. 1 and steps D and E in FIG. 4). Modulating the energy of the second working electrode relative to the second counter electrode or the third working electrode relative to the third counter electrode may be accomplished as described herein.

[0099] In embodiments where the second complex was selectively released form the second working electrode (step 310 in FIG. 1 and step C in FIG. 4), the third complex remains disposed on the third working electrode. At least a portion of the electrically conductive medium (e.g., the second solution) containing the second complex may be removed from contact with the second working electrode, for example, by removing at least a portion of the electrically conductive (e.g., second solution) from the fluidic device. In some embodiments where the second working electrode and the third working electrode are within the same channel of a fluidic device, a different electrically conductive medium (e.g., third solution) or a fresh portion of the electrically conductive medium (e.g., first solution) may be flowed into the channel to replace at least a portion of the electrically conductive medium (e.g., the second solution) that was removed from the channel after release of the second complex. In some embodiments where the second working electrode and the third working electrode are not within the same channel of the fluidic device, the electrically conductive medium or third solution may be independently present in the channel containing the third working electrode.

[0100] In embodiments where the third complex was selectively released form the third working electrode (step 310 in FIG. 1 and step E in FIG. 4), the second complex remains disposed on the second working electrode. At least a portion of the electrically conductive medium (e.g., the third solution) containing the third complex may be removed from contact with the third working electrode, for example, by removing at least a portion of the electrically conductive medium (e.g., third solution) from the fluidic device. In some embodiments whereAtorney Docket Number: 69385-727601 the third working electrode and the second working electrode are within the same channel of a fluidic device, the different electrically conductive medium (e.g., a second solution) or a fresh aliquot of the same electrically conductive medium (e.g., the first solution or third solution) may be flowed into the channel to replace at least a portion of the electrically conductive medium (e.g., third solution) that was removed from the channel after release of the third complex. In some embodiments where the third working electrode and the second working electrode are not within the same channel of the fluidic device, the electrically conductive medium (e.g., second solution) may be independently present in the channel containing the second working electrode.

[0101] In some embodiments, upon completion of step 310 (step C or E in FIG. 4), the isolation method may further include releasing the remaining complex from the respective working electrode. For example, in some embodiments, the isolation method 100 may further include selectively releasing the third complex from the third working electrode or the second complex from the second working electrode. The second complex can be released from the second electrode by modulating an energy of the second working electrode relative to the second counter electrode to release the second complex into the electrically conductive medium (e.g., second solution; step 320 in FIG. 1 and step F in FIG. 4). The third complex can be released from the third electrode by modulating an energy of the third working electrode relative to the third counter electrode to release the third complex into the electrically active medium (e.g., third solution) step 320 in FIG. 1 and step D in FIG. 4).

[0102] In some embodiments, it may be desirable to first selectively release the first complex from the first working electrode followed by simultaneously releasing the second complex and the third complex from the second working electrode and the third working electrode, respectively. In some such embodiments, the isolation method 100 may further include simultaneously modulating an energy of the second electrode relative to the second counter electrode and modulating an energy of the third electrode relative to the third counter electrode to release both the second complex into the second solution and the third complex into an electrically conductive medium. In embodiments where the second working electrode and the third working electrode are disposed within the same channel, the electrically conductive medium in which the second complex and the third complex are released into is the same (e.g., the second solution and the third solution are not physically isolated and are the same).

[0103] While FIG. 1 and FIG. 4 illustrate how isolation method 100 may be used to selectively isolate three complexes, it is understood that the number of complexes and working electrodes described in FIG. 1 and FIG. 4 is not particularly limited. For example, the first complex, the second complex, and the third complex may be a part of a plurality of molecule-Atorney Docket Number: 69385-727601 particle complexes. Each molecule-particle complex includes a molecule of interest and a particle. Molecule-particle complexes of different identities may be disposed on different working electrodes. Through individual modulation of the energy or the working electrodes, molecule-complexes of different chemical identities can be selectively released form their respective working electrodes and independently isolated from one another. As such, in some embodiments, method 100 further includes sequentially selectively releasing at least some of the plurality of molecule-particle complexes. Alternatively, the energy of groups of working electrodes may be modulated to simultaneously release groups of molecule-complexes disposed on different working electrodes.

[0104] In some embodiments, the isolation method 100 may be used to selectively isolate 2 or more, 3 or more, 6 or more, 8 or more, 10 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, 1000 or more, 5000 or more types of complexes, each type of complex having a different molecule of interest. The isolation method 100 may be used to selectively isolate 2000 or fewer, 1000 or fewer, 750 or fewer, 500 or fewer, 250 or fewer, 100 or fewer, 50 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, or 3 or fewer types of complexes, each type of complex having a different molecule of interest.

[0105] When the isolation method 100 is used to selectively isolate two or more complexes, any suitable configuration of working electrodes, counter electrodes, and channels may be used. For example, an array of all the working electrodes may be included in a single channel. Alternatively, a fluidic device may include two or more channels where one or more working electrodes are included in each channel. In some embodiments, the working electrodes may be co-planar. In some embodiments, each working electrode has an independent counter electrode. In some embodiments, all of the working electrodes share a single counter electrode. In some embodiments, the one or more counter electrodes may be coplanar to the working electrodes. In other embodiments, the one or more counter electrodes may be contacting or forming an interior face of a channel that is opposite the working electrodes.

[0106] For example, FIG. 5 is a top-down schematic view of a fluidic device 1000 that may be used to selectively isolate six complexes via the methods of the present disclosure. Fluidic device 1000 includes a first channel 600 and a second channel 700. The first channel 600 and the second channel 700 may be parallel as shown, or may be in a different configuration. Each channel includes an inlet 800 and an outlet 850. The inlets allow a fluid to enter the channel and the outlets allow fluid to exit the channel. Each channel includes three working electrodes. For example, the first channel 600 includes a first working electrode 610, a second working electrode 620, and a third working electrode 630. The second channel 700 includes a firstAtorney Docket Number: 69385-727601 working electrode 710, a second working electrode 720, and a third working electrode 730. Each working electrode may have a complex disposed thereon. For example, each working electrode may have a different complex disposed thereon. Each working electrode is electrically coupled to a power source 500 (dotted lines show direct electrical connections between power sources and electrodes as well as between electrodes). The fluidic device 1000 can include one or more counter electrodes 900, 901, 902, 903, 904, and 905. In some embodiments, the fluidic device includes a single counter electrode. In other embodiments, the fluidic device includes two or more individually controllable counter electrodes. The counter electrode or electrodes can be electrically coupled to the first, second and third working electrodes of the first channel via a fluid within the first channel. The counter electrode or electrodes can be electrically coupled to the first, second, and third working electrodes of the second channel via a second fluid within the second channel. The counter electrode or electrodes may be coplanar with the working electrodes. The fluidic device 1000 may be used as described above with regard to FIG. 4 to isolate and release the different complexes disposed on the working electrodes 610, 620, 630, 710, 720, and 730.EXAMPLES

[0107] These Examples are merely for illustrative purposes and are not meant to be overly limiting on the scope of the appended claims. Notwithstanding that the numerical ranges and parameters seting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0108] Unless otherwise noted, all parts, percentages, ratios, etc. in the examples and the rest of the specification are by weight.

[0109] The following abbreviations may be used in the following examples and / or other places in this disclosure: mL = milliliter; L = liter; LPM = liters per minute; m = meter, mm = millimeter, min = minutes; s = seconds; cm = centimeter, pm = micrometer, g = gram, min = minute, s = second, h = hour, °C = degrees Celsius, °F = degrees Fahrenheit; wt-% = weight percent; M = molar; pM = micromole; mM = millimolar; and DI water = deionized water.Attorney Docket Number: 69385-727601

[0110] Table 1 is a materials table giving a list of components used in the Examples and their associated vendor source, and abbreviation.Example 1

[0111] The purpose of Example 1 was to demonstrate the ability to site-specifically release oligomer-particle complexes from a synthesis site without imparting damage to the oligonucleotide or incidentally releasing the oligonucleotide from the particle.

[0112] Identical oligonucleotides were synthesized on silica particles adhered to different platinum-coated working electrodes arranged on a glass slide of a fluidic device. Upon synthesisAtorney Docket Number: 69385-727601 completion, two different methods were used to isolate the synthesized DNA. In the first method, the particle-DNA complexes were selectively released from some of the platinum electrodes. In the second method, the DNA on the DNA-particle complexes on the remaining electrodes was cleaved from the particles while the DNA-particle complexes were still adhered to the electrodes. The yield and quality of the DNA from the two methods were compared.

[0113] Custom substrates for synthesizing oligonucleotide were created using platinum- coated laser-patterned glass slides as electrodes with silica-based microparticles adhered to the surfaces on which DNA was synthesized. Each slide included two channels to allow for the flow of reagents and each channel contained three working electrodes. The silica microparticles were adhered to each electrode prior to DNA synthesis. The silica microparticles were silanized with APTES. These slides were placed into a flow cell that enabled reagents to flow under the control of a commercial DNA synthesizer and power to be supplied to the electrodes on the slide.

[0114] An 1 Imer (5'-TGC-ATG-CAT-TT-3') was synthesized on all six electrodes on each slide using standard phosphoramidite chemistry. The oligo synthesis was initiated by coupling dT phosphoramidite to the amine on the surface to form a covalent link to the APTES followed by a dT-linker phosphoramidite, which contains a base-labile succinyl moiety. Cy3 phosphoramidite solution was added in a 1 : 100 dilution to the terminal dT phosphoramidite of the sequence such that a small fraction of polynucleotides may have a Cy3 in place of the final dT to enable fluorescence visualization of the oligomers. The oligonucleotide sequence was synthesized using 3 vol-% trichloroacetic acid in dichloromethane as the deblock solution, 0.02 M iodine in 70 vol-% THF, 20 vol-% pyridine and 10 vol-% water as the oxidation solution, and 0.25 M 5-(ethylthio)-l-H-tetrazole (ETT) in acetonitrile as the activator.

[0115] Following the completion of synthesis, the oligonucleotide-coated silica particles in one channel of the slide were released individually from each of the three electrodes using alternating -5 V and 5 V pulses at 1 second intervals for 25 seconds total in the presence of 2 M triethylammonium acetate. The released particle-oligo complexes from each sample were individually collected and rinsed using an in-line filter. The channel was then flushed with water and dried under a flow of inert gas. This process of selectively dislodging particle-DNA complexes was repeated for each electrode in the first channel until the particle-DNA complexes from each of the three electrodes were separately dislodged and collected. This process of selectively dislodging particle-DNA complexes was independent for each electrode, which means that during the flush after the first electrode, the particle-DNA complexes on the other electrodes were still attached to the electrodes.Atorney Docket Number: 69385-727601

[0116] The parti cle-oligo complexes collected in individual filters were then exposed to AMA for 4 minutes at room temperature to cleave the succinyl linker and release the oligonucleotide from the particle into solution. The particles remained in the syringe filter while each AMA solution containing the oligonucleotides was removed. Deprotection of the oligonucleotides continued in AMA for 15 min at 60 °C. Samples were dried under vacuum and resuspended in 50 microliters of liquid chromatography -mass spectrometry (LCMS) grade water.

[0117] As a control to monitor the quality and yield obtained from the selective isolation of the DNA-particle process, the oligonucleotides synthesized on the three electrodes in the second channel of each slide were released together in a pool from the particles still adhered to the electrodes. This was done by flooding the channel with AMA to cleave the succinyl linker while the particles remained adhered to the conductive surface. The AMA solution containing oligonucleotides from all three electrodes was collected from the channel and the sample processing steps described above (deprotection, drying, and resuspension) were done.

[0118] A total of four samples were collected. Three samples were collected using the selective dislodgment of the oligonucleotide-particle complex from the three electrodes (El, E2, and E3) in the first channel (Cl) (sample names Exl-Cl-El; Exl-Cl-E2; and Exl-Cl-E3). One sample was collected from the simultaneous cleavage of the DNA while the particles were still adhered to the electrodes (All Es) in the second channel (C2; sample name is Exl-C2-All Es).

[0119] Each individual oligonucleotide sample was evaluated for quality and quantity on a binary pump Agilent HPLC / Sciex X500B mass spectrometer. The buffers used for the analysis include 100 mM HFIP and 15 mM DIPEA in water (Buffer A) and 100 mM HFIP in methanol (Buffer B). The obtained total ion chromatograms indicated that each main peak for each sample had the correct exact mass of 3320.583(FIG. 6).

[0120] Product yields were calculated from the integrated peak areas of the A260 chromatograms using standard curves and are reported per electrode area on which the particles were adhered. Average stepwise yield (ASWY) was calculated using the percent of full length product (FLP) compared to truncation and known side-products that were detected by mass spectrometry. Yields of the full-length oligonucleotide product were obtained using a diode array detector on a liquid chromatography system. Table 2 shows the results. These data show reproducibility between DNA obtained by selectively releasing the DNA-particle complexes from the different electrodes of the first channel. Additionally, the ASWY and product yields of DNA obtained from selectively isolating the DNA-particle complexes prior to cleavage wasAtorney Docket Number: 69385-727601 comparable to the DNA obtained from standard AMA-based cleaving while the particles were still adhered to the electrodes.

[0121] Table 2Example 2

[0122] The purpose of Example 2 was to determine if cross-contamination or carry-over occurs between different synthesis sites (working electrodes) within a single fluidic channel that results from the selective isolation method.

[0123] In this example, oligonucleotides were synthesized on particles disposed on two electrodes (El and E3) separated by a blank middle electrode (E2) within a single channel. Two channels having this configuration were tested. The blank electrode E2 was monitored for carryover contamination to demonstrate that adjacent electrodes do not contaminate each other.

[0124] The same substrates, flow cell, and DNA synthesizer used in Example 1 were used in Example 2. An 1 Imer (5’-TGC-ATG-CAT-TT-3’) was synthesized on all four electrodes (El and E3 of the two channels) using standard phosphoramidite chemistry. The oligo synthesis was initiated by coupling dT phosphoramidite to the amine on the surface to form a covalent link to the APTES, followed by a dT-linker phosphoramidite, which contains a base-labile succinyl moiety. Cy3 phosphoramidite solution was added in a 1 : 100 dilution to the terminal dT phosphoramidite of the sequence such that a small fraction of polynucleotides may have a Cy3 in place of the final dT to enable fluorescence visualization of the oligomers. This sequence was synthesized using an electrochemical deblock solution of 20 mM hydroquinone, 20 mM tetrachloro-benzoquinone, 50 mM tetrabutylammonium hexafluorophosphate, and 2.5 vol-% methanol in acetonitrile. The deblock was performed by applying 2 volts to each electrode used for synthesis for a total of 15 seconds followed by a 20 second dwell. Otherwise, standard chemical synthesis with 0.02 M iodine in 70 % THF / 20 % pyridine / 10 % water as the oxidation solution, and 0.25 M ETT in ACN as the activator was performed as in Example 1.Atorney Docket Number: 69385-727601

[0125] Following the completion of synthesis, the oligo-coated silica particles in the first channel of the slide were released from each of the electrodes in the channel individually as described in Example 1. The DNA was cleaved from the particles similar to Example 1.

[0126] As a control to monitor the quality and yield of oligonucleotides obtained from the selective dislodgement process, the oligonucleotides synthesized on the two electrodes in the second channel were directly released from the particles still adhered to the surface using the methods described in Example 1.

[0127] A total of four samples were collected. Three samples were collected using the selective dislodgment of the oligonucleotide-particle complex from the three electrodes (El, E2, E3) in the first channel (Cl; sample names = Ex2-Cl-El; Ex2-Cl-E2; and Ex2-Cl-E3). One sample was collected from the simultaneous cleavage of the DNA while the particles were still adhered to the electrodes (All Es) in the second channel (C2; Ex2-C2-All Es).

[0128] Each individual oligonucleotide sample was evaluated for quality and quantity using the methods described in Example 1. The obtained total ion chromatograms are shown in FIG. 7, with each main peak having the correct exact mass of 3320.583 ASWYs and full product yields per unit area were calculated according to Example 1. Table 3 shows the results. These data show oligonucleotides can be site-specifically synthesized and released from specific electrodes in a controlled manner, as shown by prominent FLP DNA peaks only on El and E3 but not E2 of the first channel (FIG. 7). The ASWY and product yield data from Cl-El and C1-E3, was similar to C2-A11 Es. This suggests that the selective dislodgement process does not have a significant negative impact on the quality or yield of DNA.

[0129] Table 3Example 3

[0130] Similar to Example 2, the purpose of Example 3 was to determine if crosscontamination or carry-over occurs between different synthesis sites (working electrodes) within a single fluidic channel that results from the selective isolation method.Atorney Docket Number: 69385-727601

[0131] Similar to Example 2, in this example, oligonucleotides were synthesized on particles disposed on two electrodes (El and E3) separated by a blank middle electrode (E2) within a single channel. The blank electrode E2 was monitored for carryover contamination to demonstrate that adjacent electrodes do not contaminate the products from El and E3. In contrast to Example 2 where the oligonucleotide sequence isolated form Cl-El and C1-E3 was the same sequence, in Example 3 the oligonucleotide sequence isolated form Cl-El, and C1-E3 was different.

[0132] The same substrates, flow cell, and DNA synthesizer used in Example 1 were used in Example 2. Additionally, the same oligonucleotide synthesis methods, including the electrochemical deblock, as described in Example 2 were used to synthesize the oligonucleotide sequence 5'-TTT-TTT-TTT-T-3 ' on the first electrode (El) and the oligonucleotide sequence 5'- CCC-CCC-CCC-T-3' on the third electrode (E3). Following the completion of synthesis, the oligo-coated silica particles were individually released from each of the electrodes as described in Example 1. The DNA was then cleaved from the particles similar to Example 1. A total of three samples were collected (El, E2, and E3).

[0133] The DNA from each sample was evaluated for quality and quantity using LCMS as described in Example 2. The resultant total ion chromatograms are shown in FIG. 8, with each main peak having the correct exact mass of 2978.501 for the oligonucleotide sequence isolated form El and 2539.458 for the oligonucleotide sequence isolated from E3. ASWYs and full product yields per unit area were calculated according to Example 1. Table 4 shows the results. These data show oligonucleotides can be site-specifically synthesized on particles on different working electrodes and the particle-oligonucleotide complexes can be released from the working electrodes in a controlled manner, as shown by prominent FLP peaks from the El and E3 sample but not the E2 sample.

[0134] Table 4Example 4

[0135] Example 4 describes one example of a manufacturing process that can be used to make the flow cells described herein. A glass slide was used as the base layer for the fluidicAtorney Docket Number: 69385-727601 device. A titanium adhesion layer as described herein was then coated onto the glass slide. A platinum conductive layer was then coated onto the adhesion layer. A laser was used to ablate the conductive layer, thereby patterning the electrodes in the conductive layer. A Kapton stencil was placed on top of the slide with cutouts for the area to be patterned with particles. The slide was then plasma treated to create a hydrophilic area for deposition of the particles. A micropipet was used to place a 4 pL suspension of 10 pm silica porous microparticles (pore size of 100 nm) in water and 2% sodium silicate onto the slide. The slide was then heated to 250 °C for one hour. Mild heat treatment was used to avoid forming glass-glass bonds between the slide and the silica particles. Lastly, the slide was treated with gaseous APTES to functionalize the surface with an amine for DNA synthesis. FIG. 9 is a schematic of a cross-section of an electrode functionalized with silica microparticles manufactured according to the process described in Example 4.Computer Systems

[0136] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 10 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, controlling the manufacturing process of a fluidic device, modulating the energy of specific electrodes within a fluidic device, or controlling synthesis of one or more oligonucleotides. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0137] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and / or data network. The networkAtorney Docket Number: 69385-7276011530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0138] The CPU 1505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0139] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0140] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additional data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0141] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0142] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.Attorney Docket Number: 69385-727601

[0143] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[0144] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0145] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such 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 may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readableAtorney Docket Number: 69385-727601 media therefore 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, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. 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.

[0146] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0275] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, control the manufacturing process of a fluidic device, modulate the energy of specific electrodes within a fluidic device, or control synthesis of one or more oligonucleotides.

[0147] The complete disclosure of all patents, patent applications, and publications cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The systems, devices, and methods described herein may not be limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

Attorney Docket Number: 69385-727601CLAIMSWhat is claimed is:

1. A method for selectively isolating a molecule, the method comprising:(a) forming a complex comprising (i) the molecule and (ii) a particle, wherein the particle is disposed on a working electrode, and wherein the working electrode is electrically coupled to a counter electrode via a solution;(b) modulating an energy of the working electrode relative to the counter electrode to release the particle from the working electrode into the solution; and(c) cleaving the molecule from the particle.

2. The method of claim 1, wherein the modulating the energy of the working electrode comprises applying a potential to the working electrode relative to the counter electrode.

3. The method of claim 2, wherein the applying the potential to the working electrode relative to the counter electrode comprises applying one or more pulses to the working electrode.

4. The method of claim 3, wherein a pulse of the one or more pulses comprises application of a positive potential and a negative potential in any order to the working electrode.

5. The method of claim 4, wherein the negative potential is from about -100 V to about -0.1 V.

6. The method of claim 5, wherein the negative potential is from about -10 V to about -3 V.

7. The method of claim 4 wherein the positive potential is from about 0.01 V to about 100V.

8. The method of claim 7, wherein the positive potential is from about3 V to about 10 V.

9. The method of claim 3, wherein a pulse of the one or more pulses has a pulse length from about 0.1 seconds to about 60 seconds.

10. The method of claim 3, wherein each pulse of the one or more pulses has a pulse length from about 0.1 seconds to about 60 seconds.

11. The method of claim 1, wherein the modulating the energy in (b) comprises a total applied energy duration from about 0.2 seconds to about 600 seconds.Atorney Docket Number: 69385-72760112. The method of claim 1, wherein the working electrode is coupled to a surface of a fluidic device, and wherein the surface of the fluidic device further comprises one or more additional working electrodes.

13. The method of claim 12, wherein the modulating the energy in (b) results in an electrochemical reaction on the working electrode, and wherein the modulating the energy in (b) does not result in an electrochemical reaction on one or more of the additional electrodes.

14. The method of claim 1, wherein the particle comprises silica, a polymer, or a metal oxide, or any combination thereof.

15. The method of claim 1, wherein the working electrode is disposed on a solid substrate.

16. The method of claim 1, wherein the working electrode is formed by a coating on a solid substrate.

17. The method of claim 16, wherein the solid substrate comprises an inert, nonconductive solid.

18. The method of claim 1, wherein the solution comprises an electrolyte.

19. The method of claim 1, wherein the solution has an ionic conductivity of 0.0001 S / m to 100 S / m.

20. The method of claim 1, wherein the solution comprises (i) water, an organic solvent, or both and (ii) an inorganic salt, an organic salt, or both.

21. The method of claim 1, wherein the particle has a diameter from about 0.001 pm to about 100 pm.

22. The method of claim 1, wherein the particle has a diameter from about 1 pm to about 50 pm.

23. The method of claim 1, wherein the particle has a diameter from about 5 pm to about 15 pm.

24. The method of claim 1, wherein (b) occurs in a fluidic device, and wherein the fluidic device further comprises one or more additional particles.Atorney Docket Number: 69385-72760125. The method of claim 22, wherein an average diameter of the particle and the one or more additional particles is from about 0.001 pm to about 100 pm.

26. The method of claim 25, wherein the average diameter of the particle and the one or more additional particles is from about 1 pm to about 50 pm.

27. The method of claim 25, wherein the average diameter of the particle and the one or more additional particles is from about 5 pm to about 15 pm.

28. The method of claim 1, wherein the particle is porous.

29. The method of claim 28, wherein the particle has an average pore diameter from about 1 A to about 2000 A.

30. The method of claim 28, wherein the particle has an average pore diameter from about 500 A to about 1500 A.

31. The method of claim 22, wherein the particle and the one or more additional particles are porous, and wherein an average pore diameter of the particle and the one or more additional particles is from about 1 A to about 2000 A.

32. The method of claim 31, wherein the average pore diameter of the particle and the one or more additional particles is from about 500 A to about 1500 A.

33. The method of claim 12, wherein the working electrode is individually addressable from one or more of the additional working electrodes.

34. The method of claim 33, wherein each working electrode of the additional working electrodes is individually addressable.

35. The method of claim 12, wherein the method further comprises (d) forming an additional complex comprising (i) an additional molecule and an additional particle, wherein the additional particle is disposed on an additional working electrode of the additional working electrodes, wherein the additional working electrode is electrically coupled to an additional counter electrode.

36. The method of claim 35, further comprising (e) modulating an energy of the additional working electrode relative to the additional counter electrode to release the additional complex from the additional working electrode into the fluidic device.Atorney Docket Number: 69385-72760137. The method of claim 35, wherein the additional working electrode and the additional counter electrode are electrically coupled via the solution.

38. The method of claim 35, wherein the additional working electrode and the additional counter electrode are electrically coupled via additional solution.

39. The method of claim 35, wherein the first molecule is different from the additional molecule.

40. The method of claim 36, wherein the first complex and the additional complex comprise a part of a plurality of molecule particle complexes, and wherein the method further comprises sequentially selectively releasing at least a portion of the plurality of molecule particle complexes.

41. The method of claim 40, wherein the plurality of molecule particle complexes comprise at least 50 molecule particle complexes.

42. The method of claim 41, wherein the at least 50 molecule particle complexes are released within a time period of no more than 30 minutes.

43. The method of claim 40, wherein the plurality of molecule particle complexes comprise at least 90 molecule particle complexes.

44. The method of claim 43, wherein the at least 90 molecule particle complexes are released within a time period of no more than 30 minutes.

45. The method of claim 1, wherein prior to (a), the particle is disposed on the working electrode by heat treatment, and wherein the heat treatment does not exceed 350 °C.

46. The method of claim 1, wherein prior to (a), the particle is disposed on the working electrode by heat treatment, and wherein the heat treatment does not exceed 300 °C.

47. The method of claim 1, wherein the molecule comprises a polynucleotide strand.

48. The method of claim 1, wherein the molecule comprises a deoxyribonucleic acid (DNA) strand.

49. The method of claim 1, wherein the molecule comprises a ribonucleic acid (RNA) strand.

50. The method of claim 1, wherein the particle comprises a silica microparticle.Atorney Docket Number: 69385-72760151. The method of claim 1, further comprising collecting the released complex in a container.

52. The method of claim 35, further comprising collecting the released complex from (b) and the released additional complex from (e) in separate containers.

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