Background

[0001 JDigital microfluidic (DMF) devices may be used to perform operations on volumes of fluid, such as the manipulation of fluid droplets to facilitate handling and testing of various fluids on a small scale. Such devices may be used for a variety of purposes and industries.

Brief Description of the Drawings

[0002]FIGs. 1A-1 G illustrate example apparatuses including digital microfluidic (DMF) devices, in accordance with examples of the present disclosure.

[0003JFIG. 2 illustrates an example DMF device, in accordance with examples of the present disclosure.

[0004JFIG. 3 illustrates an example apparatus including a DMF device and coupled circuitry, in accordance with examples of the present disclosure.

[0005]FIGs. 4A-4J illustrate operation of an example DMF device, in accordance with examples of the present disclosure.

[0006JFIG. 5 illustrates an example functionalized magnetic bead and operation thereof in a DMF device, in accordance with examples of the present disclosure. [0007]FIGs. 6A-6B illustrate other example DMF devices, in accordance with examples of the present disclosure.

[0008JFIG. 7 illustrates an example method for performing cell-free synthesis of a protein using a DMF device, in accordance with examples of the present disclosure. [0009JFIG. 8 illustrates an example device including non-transitory computer- readable storage medium, in accordance with examples of the present disclosure.

Detailed Description

[0010] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure may be practiced. It is to be understood that other examples may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0011 JDigital microfluidic (DMF) devices may be used to perform large numbers of chemical processing operations on different fluids in parallel by providing digitized movement of reaction fluids throughout the DMF devices. The digitized movement of reaction fluids may be achieved using a plurality of electrodes that form part of or are coupled to the DMF device, and which provide an electric field to drive the flow of reaction fluids as fluid droplets. The electrodes may be arranged into a two-dimensional array and are individually addressable by circuitry, sometimes herein referred to as “selective actuation”, to provide the digitized and controlled flow of picoliter to microliter sized fluid droplets of reaction fluids by drawing the fluid droplets toward an addressed electrode. As the respective electrode is addressed, the addressed electrode provides an electric field within the DMF device and/or onto the reaction fluid, and due to a charge of the reaction fluid, a fluid droplet of the reaction fluid is directed along a microfluidic path. The movement of the fluids within the DMF device may be used to move, mix, and/or split fluid droplets of reaction fluids into two respective smaller fluid droplets, among other uses, and to drive a chemical processing operation thereon. [0012]ln some examples, DMF devices may be designed to perform chemical processing operations on chemical components to synthesize proteins. In many instances, it may be useful to generate proteins exhibiting particular behavior and/or functions. Furthermore, proteins may be used for a variety of purposes, such as for medical treatments, sensors, food additives, among other purposes. Proteins may be expressed in vivo, where a nucleic acid sequence that encodes a protein is introduced into an organism, such as bacteria, and cells of the organism express the protein using the nucleic acid sequence. In some instances, proteins are expressed in vitro via a cell-free synthesis process by mixing nucleic acids with reagents containing enzymatic machinery and building blocks, similar to cells, for transcribing and/or translating the protein from the nucleic acids. As used herein, cell-free synthesis refers to or includes an in vitro protein synthesis that is performed without the use of living cells. With both in vitro and cell-free synthesis of proteins, the protein synthesized may not be pure, which may result in performing additional actions to purify the protein so that the function of the protein is not altered by contaminating substances. Both the protein synthesis and the purification processes may be time consuming and involve manual labor.

[0013]Examples of the present disclosure are directed to a DMF device that performs protein synthesis and purification within the DMF device and in an automated and integrated manner. The protein is synthesized by selectively actuating electrodes of a two-dimensional array of electrodes to merge a nucleic acid template with reagents containing enzymatic machinery and building blocks for transcribing and translating a protein from the nucleic acid template, and to provide an isolated thermal zone to drive the cell-free synthesis of the protein within the DMF device. The protein is then purified by using functionalized magnetic beads and a magnet associated with the DMF device to capture and isolate the protein from other components within fluids of the DMF device and wash the protein. By integrating the protein synthesis and purification on the DMF device, the DMF device reduces the laborious process of manually handling different reaction fluids and performing different synthesis and purification processes. More specifically, as protein synthesis and purification is performed on the DMF device, purified proteins may be generated within the DMF device without or with minimal manual handling of reaction fluids and/or user manual operation of the DMF device. Further, as the protein is purified, the protein may be assayed for functionalities, such as enzymatic activity. The protein may be assayed on the DMF device using a sensor of the DMF device or an external optical sensing device, or off-device.

[0014]Example DMF devices comprise a housing including an interconnected chamber that defines a plurality of microfluidic paths, a plurality of fluidic inlets to input different reaction fluids, and a magnetic unit coupled to and disposed along a portion of the interconnected chamber. The different reaction fluids may be inserted into the DMF device via the fluidic inlets, and via the electrodes, are formed into fluid droplets of reaction fluids and selectively carried through respective ones of the plurality of microfluidic paths to drive cell-free protein synthesis and purification. Example reaction fluids contain or include functionalized magnetic beads, an enzyme reagent, a nucleic acid template that encodes a protein, a wash buffer, and an elution buffer. The magnetic beads may be functionalized to bind to the protein based on size and/or chemical properties of the protein. The selectively movement of the fluid droplets of the reaction fluids may include sequential movement of respective fluids to synthesize the protein, isolate and purify the protein, and, optionally, flow the protein to a region of the DMF device for interrogation.

[0015]As used herein, a chamber refers to or includes an enclosed and/or semienclosed region of the DMF device, which may be formed of an etched or micromachined portion (e.g., negative space forming a conduit in a substrate or substrates) and which may be used to perform chemical processing on fluids therein. An interconnected chamber refers to or includes a chamber that defines a plurality of microfluidic paths via use of the 2D array of electrodes. In some examples, the fluid droplet of a reaction fluid may include a volume of about 1 microliter (pL) or less, such as a volume of between about 0.1 pL and about 1 pL, about 0.25 pL and about 1 pL, about 0.5 pL and about 1 pL, about 0.5 pL and about 0.75 pL, about 0.25 pL and about 0.75 pL, or about 0.1 pL and about 0.5 pL, among other ranges. In some examples, the fluid droplet may be larger, such as on a nanoliter (nL) scale or between about 0.1 nL and about 0.5 nL. A channel refers to or includes a path through which a fluid or semi-fluid may pass, which may allow for transport of volumes of fluid on the order of pL, nanoliters, picoliters, or femtoliters. As used herein, a well, such as a reaction fluid well, refers to or includes a column capable of storing a volume of fluid. In some examples, the well may store a volume of fluid that includes more than one droplet of fluid, such as at least two fluid droplets of a reaction fluid. In some example, a well may store a volume of fluid in a range between about 1 pL and about several milliliters (mL) of fluid. In some examples, the well may store a volume of fluid between about 1 pL and about 1 mL, about 1 pL and about 500 pL, about 1 pL and about 50 pL, about 1 pL and about 10 pL, or about 1 pL and about 5 pL, among other volume ranges. A reservoir refers to or includes a column where fluid collects, such as the waste fluid. In some examples, a reservoir may collect a larger volume of fluid than a well may store.

[0016]A reaction fluid refers to or includes fluid containing substances, molecules, mixtures, and/or other components used to drive a biochemical reaction, such as for cell-free synthesizing, purifying, and/or assay a protein. A fluid droplet of a reaction fluid, as used herein, refers to or includes a discrete portion of fluid (e.g., a liquid), which may be surrounded by a carrier fluid. A carrier fluid refers to or includes fluid that flows through portions of the DMF device and which carries solid or fluid particles, such as fluid droplets of the reaction fluids. As an example of a fluid droplet of the reaction fluid, an immiscible fluid, such as an aqueous solution, is surrounded by an oil phase. Fluid droplets of reaction fluids may be formed from a fluid packet of the reaction fluid. A fluid packet of the reaction fluid refers to or includes a volume of fluid that is larger than a fluid droplet of the reaction fluid.

[0017]A functionalized magnetic bead refers to or includes a bead having magnetic properties and/or is otherwise capable of being attracted to or repelled by a magnetic field. A bead refers to or includes a material formed in a three- dimensional shape, such as a sphere, an ellipsoid, oblate spheroid, and prolate spheroid shapes. As further described below, the functionalized magnetic beads may be between 1 micrometer (pm) and 20 millimeter (mm) in diameter as non- limiting examples. A magnetic unit refers to or includes circuitry and/or a physical structure that causes or outputs a magnetic field.

[0018]Some examples are directed to an apparatus comprising a DMF device and circuitry. The DMF device including a housing including an interconnected chamber and with a two-dimensional array of electrodes couplable to the interconnected chamber, a plurality of fluidic inlets fluidically coupled to the interconnected chamber to input a plurality of reaction fluids including a functionalized magnetic bead, an enzyme reagent, and a nucleic acid template that encodes a protein for cell-free protein synthesis, and a magnetic unit coupled to the interconnected chamber. The circuitry to selectively actuate electrodes of the two-dimensional array of electrodes and the magnetic unit to cause movement of fluid droplets of the plurality of reaction fluids and to provide a thermal zone within a first portion of the interconnected chamber.

[0019] In some examples, the circuitry is to selectively actuate the electrodes and the magnetic unit to move the fluid droplets of the plurality of reaction fluids and provide the thermal zone to synthesize the protein and bind the protein to the functionalized magnetic bead, and move the protein bound to the functionalized magnetic bead to a second portion of the interconnected chamber to purify the protein, wherein the magnetic unit is disposed along the second portion.

[0020] In some examples, the DMF device further includes a sensor disposed with another portion of the interconnected chamber.

[0021 ] In some examples, the housing further includes a base substrate and a top substrate, the interconnected chamber including a bottom surface defined by the base substrate and a top surface defined by the top substrate and the two-dimensional array of electrodes being couplable to the base substrate. And, a carrier fluid is contained within the interconnected chamber.

[0022]ln some examples, the DMF device further includes a plurality of reaction fluid wells to contain the plurality of reaction fluids, wherein the plurality of reaction wells are fluidically coupled to the plurality of fluidic inlets.

[0023] In some examples, the apparatus further includes a region to collect the protein, wherein the region is selected from: a collection well fluidically coupled to the interconnected chamber; a substrate disposed within a well fluidically coupled to the interconnected chamber; and another portion of the interconnected chamber.

[0024] In some examples, the apparatus further includes a waste reservoir fluidically coupled to the interconnected chamber.

[0025]Some examples are directed to an apparatus comprising a DMF device, a two-dimensional array of electrodes, a magnetic unit, and circuitry. The DMF device including a housing including an interconnected chamber that defines a plurality of microfluidic paths, and a plurality of fluidic inlets fluidically coupled to the interconnected chamber to input a plurality of reaction fluids including a functionalized magnetic bead, an enzyme reagent, and a nucleic acid template that encodes a protein. The apparatus further includes a magnetic unit coupled to and disposed along the interconnected chamber, and a two-dimensional array of electrodes coupled to and disposed along the interconnected chamber. The apparatus further includes circuitry communicatively coupled to the magnetic unit and the two-dimensional array of electrodes to selectively actuate electrodes of the two-dimensional array of electrodes to move fluid droplets of the plurality of reaction fluids along respective ones of the plurality of microfluidic paths within the interconnected chamber and to generate a thermal zone within the interconnected chamber associated with a microfluidic path of the plurality of microfluidic paths, and selectively actuate the magnetic unit to move the functionalized magnetic bead toward the magnetic unit to facilitate cell-free synthesis and purification of the protein.

[0026] In some examples, the apparatus further includes a driving instrument including the circuitry and the magnetic unit, the driving instrument to receive the DMF device.

[0027] In some examples, the DMF device further includes an interrogation region, and the apparatus further includes a sensor to interrogate the protein disposed in the interrogation region.

[0028]ln some examples, the DMF device further includes a plurality of reaction fluid wells to contain the plurality of reaction fluids and fluidically coupled to the plurality of fluidic inlets, the plurality of reaction fluids being selected from: a first reaction fluid containing the enzyme reagent including a polymerase; a second reaction fluid containing the nucleic acid template that encodes the protein; a third reaction fluid containing the functionalized magnetic bead; a wash buffer fluid; an elution buffer fluid; and a combination thereof.

[0029] In some examples, the interconnected chamber contains a carrier fluid, and the circuitry is to selectively actuate electrodes of the two-dimensional array of electrodes to form the fluid droplets as surrounded by the carrier fluid.

[0030]Some examples are directed to a method comprising flowing a first fluid droplet of a first reaction fluid along a microfluidic path within an interconnected chamber of a DMF device via application of electrowetting forces by a two- dimensional array of electrodes, the first reaction fluid containing a nucleic acid template that encodes a protein, and merging the first fluid droplet of the first reaction fluid with a second fluid droplet of a second reaction fluid containing an enzyme reagent to form a first merged fluid droplet. The method further comprises generating a thermal zone in the interconnected chamber proximal to the first merged fluid droplet via actuation of an electrode of the two-dimensional array of electrodes to drive cell-free synthesis of the protein within the first merged fluid droplet, merging the first merged fluid droplet containing the protein with a third fluid droplet of buffer fluid containing a functionalized magnetic bead to form a second merged fluid droplet, wherein the protein is to bind to the functionalized magnetic bead, and applying a magnetic field to the second merged fluid droplet via a magnetic unit disposed along the interconnected chamber, and directing molecules in the second merged fluid droplet not bound to the functionalized magnetic bead to a waste reservoir to purify the protein. The method further comprises flowing the purified protein along a second microfluidic path of the interconnected chamber to a portion of the DMF device. [0031 ] In some examples, the method further includes flowing a fourth fluid droplet containing a chemical substrate to the portion with the purified protein, and interrogating the portion for a product or flowing the fourth fluid droplet containing the chemical substrate and the purified protein to another portion of the DMF device and interrogating the other portion for the product using at least one of a sensor disposed in the interconnected chamber and an optical sensing device coupled to the DMF device.

[0032] In some examples, method further includes repeating cycles of turning off the magnetic field, allowing the functionalized magnetic bead to mix with additional fluid droplets containing wash buffer fluid, and moving the additional fluid droplets containing the wash buffer fluid and respective unbound molecules of the second merged fluid droplet to the waste reservoir. The method further including separating the purified protein from the functionalized magnetic bead by flowing a fourth fluid droplet of elution buffer fluid and merging the fourth fluid droplet of elution buffer fluid with the second merged fluid droplet to displace the purified protein from the functionalized magnetic bead.

[0033]Turning now to the figures, FIGs. 1A-1 G illustrate example apparatuses including DMF devices, in accordance with examples of the present disclosure. The apparatuses 130 each include a DMF device 100 and circuitry 103.

[0034] As shown in FIG. 1A, an example apparatus 130 includes a DMF device 100 that comprises a housing 102 including an interconnected chamber 104 and with two-dimensional (2D) array of electrodes 106 coupled to the housing 102. The housing 102 may include substrates, with the interconnected chamber 104, among other components, formed by and/or between the substrates as etched or micromachined portions. The etched or micromachined portions forming the interconnected chamber 104, and optionally additional chambers, wells, and/or channels may be a height in the range of about 10 pm to about 2 mm. Each substrate, such as the top and base substrates 102-1 , 102-2 illustrated by FIG. 1 B, may be formed of a plurality of different materials which are in layers, as further described herein. Referring back to FIG. 1A, in some examples, the interconnected chamber 104, and optionally other chambers, wells, and channels, may be formed by etching or micromachining processes in a substrate to form the various etched or micromachined portions. Accordingly, the chamber(s), wells, and/or channels may be defined by surfaces fabricated in the substrate(s) of the DMF device 100.

[0035]The 2D array of electrodes 106 are couplable to the housing 102 and may be disposed proximal to the interconnected chamber 104. As used herein, a 2D array of electrodes includes or refers to a plurality of electrodes which are arranged in an array in at least two directions. The interconnected chamber 104 may be used to provide the plurality of different microfluidic paths via selective actuation of respective electrodes of the 2D array of electrodes 106. In some examples, the 2D array of electrodes 106 are positioned along and/or exposed to the interconnected chamber 104. In other examples, the 2D array of electrodes 106 are coupled to the housing 102, as further described herein. Proximal, as used herein (e.g., proximal to a microfluidic path and/or the interconnected chamber 104), refers to or includes being disposed in line with a portion of the DMF device 100, such as being positioned along, above, below, and/or exposed to the portion of the DMF device 100.

[0036]As further described herein, the 2D array of electrodes 106 are to actuate to selectively move a plurality of reaction fluids along respective microfluidic paths within the interconnected chamber 104. Example electrodes include transparent electrodes, ring electrodes, linear electrodes, almost continuous electrodes, ground electrodes, and/or actuating electrodes, among others. The plurality of electrodes 106 may be the same size or different sizes. The electrodes may be formed of a conductive material, such as metal, conductive polymers, indium tin oxide (ITO), transparent conductive oxides, carbon nanotube, among other material.

[0037]As used herein, a transparent electrode refers to or includes an electrode that is transparent or semi-transparent. Use of transparent electrodes, along with a transparent lid, as further described below, may allow for a user to visually view fluid flow within the DMF device 100 while chemical operations are being performed by the DMF device 100 and/or to verify proper fluid processing is occurring. A ring electrode refers to or includes an electrode which is annulus shaped. The ring electrode(s) may be shaped to extend around a portion of the DMF device 100, such as a portion of the interconnected chamber 104. A linear electrode refers to or includes an electrode which extends in a straight line and for a sub-portion of the DMF device 100. A plurality of linear electrodes may be placed in an array along a portion of the DMF device 100 (e.g., a portion of the interconnected chamber 104), and may provide greater control of fluid flow, as compared to an almost continuous electrode, due to known electrode positions and localized resolution. An almost continuous electrode refers to or includes an electrode which extends along a portion of the DMF device 100, such as along the bottom surface or top surface of the interconnected chamber 104 (e.g., see electrode 106-5 of FIG. 1 B). An almost continuous electrode may reduce manufacturing costs, as compared to an array of linear electrodes.

[0038JA ground electrode refers to or includes an electrode that provides or establishes a connection to ground. An actuating electrode refers to or includes an electrode that is actuated (e.g., a voltage is applied thereto by coupled circuitry), and in response, generates an electric field based on a differential between the actuating electrode (e.g., the applied voltage) and ground. In some examples, ground may be provided by a ground electrode, and in other examples, ground is provided by fluid within the DMF device 100. Use of a ground electrode may provide greater control of fluid flow or formation of a fluid droplet of the reaction fluids as compared to use of fluid within the interconnected chamber 104 as ground. Using fluid as ground may reduce manufacturing costs.

[0039]The DMF device 100 further includes a plurality of fluidic inlets 110 fluidically coupled to the interconnected chamber 104. A fluidic inlet refers to or includes an inlet port, e.g., an aperture, that is fluidically coupled to the interconnected chamber 104. The plurality of fluidic inlets 110 may be used to input a plurality of reaction fluids into the DMF device 100. The plurality of reaction fluids may include or contain an enzyme reagent, a nucleic acid template, and a plurality of buffer fluids. The buffer fluids may include a buffer fluid containing the functionalized magnetic bead, a wash buffer fluid, and an elution buffer fluid. Although examples describe a buffer fluid containing a functionalized magnetic bead or a fluid droplet of a buffer fluid containing the functionalized magnetic bead, in various examples, the buffer fluid and/or fluid droplet of the buffer fluid contains a plurality of functionalized magnetic beads. [0040]The reaction fluids may include fluids used to perform cell-free protein synthesis. Cell-free protein synthesis includes the production of recombinant proteins in solution using the biomolecular enzymatic and/or translation machinery extracted from cells. The protein synthesis occurs in cell lysates rather than within cultured cells. The protein may be synthesized using a reaction fluid containing cell lysates, as further described below. The cell lysates may include cell extracts derived from prokaryotic organisms, such as E. coli, insects, mammals, among other organisms.

[0041] The reaction fluid containing the enzyme reagent may include a polymerase and/or other reagents for transcribing and translating the protein from the nucleic acid template. A nucleic acid template refers to or includes a sequence of nucleotides that encodes the protein, and optionally, other components, such as protein tags. A nucleotide refers to or includes a nucleoside and a phosphate, which serve as monomer units for forming proteins. The nucleic acid template may include a DNA or RNA template. Polymerase is an enzyme which may synthesize the protein from the nucleic acid template by copying the template using base-paring interactions. In some examples, the reaction fluid may include T2 S30™ which contains the T7 RNA polymerase to transcribe a DNA template and the biomolecular translation machinery to express the protein by translation. In some examples, the reaction containing the enzyme reagent may further include building blocks for transcription and translation, such as nucleoside triphosphate (NTP), nucleotides and/or amino acids. Other example polymerases include T3 RNA polymerase and SP6 RNA polymerase, and various combinations thereof.

[0042] In some examples, the reaction fluid containing the enzyme reagent may include a cell extract solution includes an RNA polymerase for messenger RNA (mRNA) transcription, ribosomes for polypeptide translation, transfer RNA (tRNA), amino acids, enzymatic cofactors and an energy source, and/or other cellular components for driving protein folding. The cell-free synthesis may occur using a DNA template via transcription and translation, or an RNA template via translation. Transcription includes the process by which DNA is copied to RNA. For example, a complementary single stranded mRNA is generated from the DNA template using a polymerase during transcription. Translation includes a process by which RNA is used to produce proteins. For example, from the mRNA generated by the transcription process, a protein is produced. The nucleic acid mixes and reacts with the reaction fluid including the enzyme reagent, and is incubated at a temperature to synthesize the protein (e.g., between about 30 degrees Celsius (C) and about 40 degrees C, or about 37 degrees C).

[0043]For a DNA template encoding a protein, a promoter sequence is located upstream of the gene encoding the protein to be transcribed in the DNA template. Transcription may include initiation, elongation, and termination. For initiation, the RNA polymerase (RNApol) uses specific sequences or elements to bind to the promoter sequence and to initiate transcription. Some RNApols have one sub-unit, such as those from bacteriophages like T3 and T7, and mitochondria, while other RNApols from bacteria and eukaryotes are multisubunit enzymes that use transcription factors for efficient transcription. The multimeric enzymes may be difficult to reconstitute from purified subunits. By contrast, the smaller monomeric RNApols from bacteriophages may perform transcription, including termination and release of the transcript from a DNA template, without the aid of transcription factors. Transcription factors include proteins that assist with positioning the RNApol and assist in breaking hydrogen bonds in the DNA helix. In some examples, the reaction fluid including the enzyme reagent includes the transcription factors. Downstream of the promoter sequence, the DNA template includes the sequence encoding the protein. After binding to the promoter sequence, for elongation, the RNApol breaks hydrogen bonds connecting two strands of DNA in the DNA template and then uses a single DNA strand to build an RNA strand in the 5’ to 3’ direction, adding each complementary nucleotide to the 3’ end of the strand. In RNA, the nucleotide thymine (T) is replaced by the nucleotide uracil (U). The resulting RNA strand is released from the DNA strand as a single strand during termination. The mRNA generated during transcription includes a sequence of nucleotides. A set of three nucleotides, sometimes referred to as a codon, encodes for an amino acid, a start signal for translation, or stop signal for the end of translation.

[0044]For an RNA template or from the mRNA strand generated by the translation process, the mRNA may be translated via initiation, elongation, and termination. As noted above, the reaction fluid including the enzyme reagent may contain ribosomes, tRNA, amino acids, and other components (e.g., initiation factors) which may be used for translation. A ribosome is a particle including RNA and proteins that performs translation, and which includes small (40S) and large (60S) subunits. tRNA is a molecule including anticodons, which are a sequence of three nucleotides that are complimentary to specific codons in mRNA. For initiation, the ribosome binds to the start codon of the mRNA sequence, e.g., AUG, which codes for the amino acid methionine. The tRNA carrying an anticodon recognizes the start codon and carries the amino acid methionine to the mRNA. The large subunit of the ribosome then binds to form an initiation complex. The initiation complex is a complex including the tRNA, the small subunit of the ribosome, and the large subunit of the ribosome, which may assemble into the complex (e.g., an 80S ribosome) by initiation factors. For elongation, the ribosome continues down the mRNA strand translating each codon to an amino acid and the corresponding amino acids are added by the tRNA and are linked together by peptide bounds. This continues until the ribosome reaches the stop codon for termination. Example stop codons include UAA, UAG, and UGA. As there are no tRNAs that read and recognize the stop codons to recruit an amino acid, and the ribosome recognizes that the translation process is finished and releases the protein.

[0045]The reaction fluid containing the nucleic acid template that encodes the protein may be a buffer fluid. As noted above, the nucleic acid template may include a DNA or RNA template, e.g., sequence, that encodes for the protein. For example, in response to transcription and/or translation of the nucleic acid template by the enzyme reagent(s), the protein is synthesized. For a DNA template, the template may be linear or circular.

[0046]Buffer fluids refer to or include fluids which assist in maintaining a pH within fluids, such as mitigating pH changes and/or maintaining the pH within a range. Example buffer fluids include a solution with a weak base or acid, such as a solution containing citrate, acetate, or phosphate salts. Example buffer fluids containing the functionalized magnetic beads include a phosphate buffered saline or a carbonate-bicarbonate buffer, among other buffer fluids. [0047]Some example buffer fluids include fluids containing salt such as 2-(N- morpholino)ethanesulfonic acid (MES), Bis-tris methane, 2,2’,2"-Nitrilotriacetic acid (ADA), Bis-tris propane, and/or piperazine-N,N'-Bis(2-ethanesulfonic acid) (PIPES). Other non-limiting example buffer fluids include fluids containing tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2- hydroxyethyl)amino)acetic acid (Bicine), tris(hydroxymethyl)aminomethane, or 2-amino-2-(hydroxymethyl)propane-1 ,3-diol) (Tris), N- [tris(hydroxymethyl)methyl]glycine (Tricine), 3-[N- tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), 2-[[1 ,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N- morpholino)propanesulfonic acid (MOPS), 2-Hydroxy-3- morpholinopropanesulfonic acid (MOPSO), dimethylarsenic acid (Cacodylate), 2-[(2-Amino-2-oxoethyl)amino]ethane-1 -sulfonic acid (ACES), cholamine chloride, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2- hydroxyethyl)taurine (BES), 2,2-bis(2-Hydroxyethyl) -3-amino-2-hydroxypropane sulphonic acid (DIPSO), acetamidoglycine, triethanolamine (TEA), and Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), among others. In some examples, the buffer fluids may include a tris-buffered saline, such as Tris-hydrochloric acid (HCI), sodum chrolide (NaCI) containing imidazole. [0048]As described above, the functionalized magnetic bead(s) may have functionalized surfaces to selectively bind to the synthesized protein and not bind to other molecules in the reaction fluids. The functionalized magnetic beads may be used to segregate the synthesized protein from the other molecules in the reaction fluids based on size, chemical properties, and/or combinations thereof, sometimes herein referred to as “bead-based separation of molecules”. By segregating the synthesized protein, the DMF device 100 may be used to purify the protein, as further described herein.

[0049]The functionalized magnetic bead(s) may be of a size such that the beads are capable of moving through the interconnected chamber 104. For example, the functionalized magnetic bead(s) may be between 1 pm and 20 mm in diameter as non-limiting examples. The functionalized magnetic bead(s) may be formed of, for example, glass, polymer, silica, alumina, silicon carbide, tungsten carbide iron oxide steel, silica coated metal, boron nitride, or other suitable material which is magnetic, is made with magnetic atoms, and/or includes a core with a magnetic coating. For instance, example functionalized magnetic bead(s) may consist essentially of iron oxide, a soft ferrite, a ferromagnetic material, a ferrimagnetic material, and/or combinations thereof. Non-limiting example compositions of functionalized magnetic bead(s) include iron oxide (Fe2O3), soft ferrites ranging from spinel-type ferrites (MeFe2O4) to manganese-zinc ferrite (Mn _a Zn(i- _a )Fe2O4), nickel-zinc ferrite (Ni _a Zn(i- _a )Fe2O4), and a nickel-iron alloy (Ni-Fe (80:20)), among others. The functionalized magnetic bead(s) may be spherical, such as beads, or may not be spherical, such as disk-shaped, rock or gravel-like, or other suitable shapes. The functionalized magnetic bead(s) may be monodispersed or poly-dispersed. In some examples, the functionalized magnetic bead(s) may include a core formed of a non-magnetic material and a magnetic coating, such as a tungsten carbide core and an iron oxide coating as a non-limiting example. The core may increase the density of the functionalized magnetic bead(s) and the magnetic coating provides the magnetic properties.

[0050]Example wash buffer fluid includes deionized water or another buffer fluid, such as those described above. As further described herein, the wash buffer fluid may be used to wash away molecules that are not bound to the functionalized magnetic bead.

[0051]Example elution buffer fluid includes acetonitrile, ethanol, and hexane. In some examples, the elution buffer fluid includes acetonitrile or imidazole. The elution buffer fluid may be used to elute and/or displace the bound synthesized protein from the functionalized magnetic bead.

[0052] In some examples, the protein is engineered to contain a protein tag. As used herein, a protein tag refers to or includes a functional group, molecule, or compound which is genetically grafted onto the protein. For example, the nucleic acid template may encode for the protein and the protein tag, such that the synthesized protein exhibits the protein tag and binds to the functionalize bead via the protein tag. The protein tag may be removeable by a chemical agent or enzyme. The protein tag may include an affinity tag, a solubilization tag, a fluorescence tag, a chromatography tag, or an epitope tag, among others. Non-limiting example protein tags include a histidine tag, albumin binding protein, a FLAG tag, alkaline phosphate (AP), an ALII epitope, an AU5 epitope, a bacteriophage T7 epitope, a bacteriophage V5 epitope, a fluorescent protein, such as a green fluorescent protein, horseradish perioxide, LacZ, luciferase, S- tag, protein C, protein A, protein G, strep-tag, and ubiquitin, among other tags. [0053]The apparatus 130 further includes a magnetic unit 108 coupled to and disposed along a portion of the interconnected chamber 104. The magnetic unit 108 may include a magnet that provides a magnetic field and electrical connects which may couple to circuitry, such as the circuitry 103 illustrated by FIG. 1 B. The magnetic unit 108 may form part of the DMF device 100 or part of a driving instrument including the circuitry 103.

[0054] In some examples, the magnet of the magnetic unit 108 includes an electromagnet which is selectively actuated to output a magnetic field used to attract the functionalized magnetic bead, as further described below. The electromagnet of the magnetic unit 108 may be actuated in response to an electrical signal (e.g., a voltage) applied thereto, and in response, outputs the magnetic field. The magnetic field may be subsequently deactivated or removed by removing the electrical signal.

[0055] In some examples, the magnet of the magnetic unit 108 includes a permanent magnet, and the magnetic unit 108 further includes or is coupled to a stage or other movable hardware that moves the permanent magnet to different positions. For example, the permanent magnet may be moved positions to provide a magnetic field within the interconnected chamber 104 that is sufficient to attract the functionalized magnetic bead and then moved to a position that the magnetic field within the interconnected chamber 104 is insufficient to attract the functionalized magnetic bead.

[0056]The apparatus 130 further includes circuitry 103. The circuitry 103 is communicatively coupled to the 2D array of electrodes 106 and the magnetic unit 108 to selectively actuate electrodes of the 2D array of electrodes 106 and the magnetic unit 108 to move fluid droplets of the plurality of reaction fluids along different microfluidic paths within the interconnected chamber 104. In some examples, the circuitry 103 may be supported by the housing 102. In other examples, the circuitry 103 may be supported by another device and is couplable to the DMF device 100. For example, the circuitry 103 may be external to the housing 102-1 , 102-2 and/or the DMF device 100.

[0057]ln some examples, the circuitry 103 selectively actuates electrodes of the 2D array of electrodes 106 and the magnetic unit 108 to cause movement of fluid droplets of the plurality of reaction fluids to a first portion of the interconnected chamber 104 and to provide a thermal zone within the first portion to drive cell-free synthesis and purification of the protein. In some examples, the circuitry 103 may move the fluid droplets of the plurality of reaction fluids along respective ones of a plurality of microfluidic paths to the first portion of the interconnected chamber 104 and generate the thermal zone within the first portion of the interconnected chamber 104 to synthesize the protein and bind the protein to the functionalized magnetic bead, and purify the protein bound to the functionalized magnetic bead. The thermal zone may be localized. A localized thermal zone refers to or includes a temperature generated within the portion of DMF device 100 which does not or mitigates an impact to temperatures of other portions of the DMF device 100, such that the other portions of the DMF device 100 remain at or near ambient temperature or a temperature set by generating other thermal zones. An example of the selectively flow is further illustrated herein, at least by FIGs. 4A-4J.

[0058]Example circuitry includes a processor and memory, as further described below. In other examples, the circuitry 103 includes an anisotropic conductive layer (e.g., an anisotropically decoupling layer 103-1 and the 2D array of electrodes 106 illustrated by FIG. 1 G) of the DMF device 100 which may conduct electricity in one direction, and is coupled to the 2D array of electrodes 106 and is couplable to external circuitry, such as an external processor and/or memory. Using a conductive layer on the DMF device 100 may reduce costs of the DMF device 100, which may be disposable. Use of processor and/or memory may allow for greater control of fluid flow as compared to use of external processor and/or memory. [0059]The apparatus 130 illustrated by FIG. 1A may include variations, some of which are illustrated by FIGs. 1 B-1 G. The variations may include, but are not limited to, top and base substrates forming the interconnected chamber, a base substrate and side substrates forming the housing, electrodes disposed on the top and/or base substrates of the interconnected chamber, electrodes disposed on an additional substrate couplable to the housing, fluidic inlets disposed through the top substrate or through the side substrates, among others. Each DMF devices of FIGs. 1 B-1 G include an implementation of the DMF device 100 of FIG. 1 A, including at least some of the same features and components, as illustrated by the common numbering. The common features and components are not repeated for ease of reference.

[0060]FIGs. 1 B-1 C illustrate an example implementation of apparatus 130 including the DMF device 100 of FIG. 1A. FIG. 1 B is a cross-sectional view of the interconnected chamber 104 of the example implementation of the DMF device 100 and FIG. 1 C is a top view of the example implementation. As shown, the housing 102-1 , 102-2 of the DMF device 100 includes a base substrate 102- 1 and a top substrate 102-2. In some examples, the top substrate 102-2 may form or include a lid of the DMF device 100. In some examples, the top substrate 102-2, or a portion thereof, may be transparent. In some examples, the top substrate 102-2 may be transparent, and, in other examples, both the top and base substrates 102-1 , 102-2 are transparent. A transparent substrate(s) (and optionally electrodes) may allow for optical monitoring of fluid flow and/or chemical operations within the DMF device 100 by a user, which may be used to visually verify the DMF device 100 is functioning properly. [0061]ln some examples, the interconnected chamber 104 includes a bottom surface 109 defined by the base substrate 102-1 and a top surface 107 defined by the top substrate 102-2. A bottom surface of the interconnected chamber refers to or includes a floor or lower surface of the chamber with respect to gravity. A top surface of the interconnected chamber refers to or includes a ceiling or overhead surface of the chamber with respect to gravity.

[0062]The plurality of electrodes 106-1 , 106-2, 106-3, 106-4, 106-5 of the DMF device 100 may be arranged in a 2D array (herein generally referred to as “the 2D array of electrodes 106” for ease of reference) and are coupleable to the base substrate 102-1 . In some examples, and as shown by FIG. 1 B, the 2D array of electrodes 106 are disposed on or within the base substrate 102-1 . In some examples, the 2D array of electrodes 106 may extend level with or extrude beyond the bottom surface 109 or top surface 107 of the interconnected chamber 104, such that the electrodes may be in contact with fluids contained in the interconnected chamber 104. However, examples are not so limited, and the 2D array of electrodes 106 may be disposed within the base substrate 102-1 and may not be exposed to fluids in the interconnected chamber 104, may have a coating disposed on the 2D array of electrodes 106 and/or may be disposed in another substrate, such as substrate 111 illustrated by FIG. 1 G.

[0063] Referring to FIG. 1 B, in some examples, the electrodes of the 2D array of electrodes 106 include actuating electrodes 106-1 , 106-2, 106-3, 106-4 and a ground electrode 106-5. The actuating electrodes 106-1 , 106-2, 106-3, 106-4 may be disposed on or within the base substrate 102-1 and the ground electrode 106-5 may be disposed on or within the top substrate 102-2. Use of a ground electrode 106-5 with the plurality of actuating electrodes 106-1 , 106-2, 106-3, 106-4 may allow for greater control of fluid flow and/or formation of fluid droplets of the reaction fluids as compared to using fluid control without the ground electrode 106-5. For example, to provide flow, the charge from the actuating electrodes 106-1 , 106-2, 106-3, 106-4 may go to ground. Without the use of the ground electrode 106-5, a stray charge may accumulate in the DMF device 100, which produces an electric field and causes forces on fluid therein. [0064]Although FIG. 1 B illustrates the ground electrode 106-5 as a single electrode (e.g., an almost continuous electrode), examples are not so limited. In some examples, a plurality of ground electrodes may be disposed on or within the top substrate 102-2. In other examples, all of the electrodes of the 2D array of electrodes 106 are actuating, and no ground electrodes are used. In some examples, at different points in time, respective electrodes of the 2D array of electrodes 106 may be floating or set at ground, such as when the respective electrodes are not being used to draw the fluid along the microfluidic path. [0065]As further illustrated by and referring to FIG. 2, the DMF device 200 may include a plurality of reaction fluid wells 222-1 , 222-2, 222-3, 222-4, 222-5 (herein generally referred to as “the reaction fluid wells 222” for ease of reference). The reaction fluid wells 222 may contain the plurality of reaction fluids 220-1 , 220-2, 220-3, 220-4, 220-5 (herein generally referred to as “the reaction fluids 220” for ease of reference) and are fluidically coupled to the plurality of fluidic inlets 210-1 , 210-2, 210-3, 210-4, 210-5.

[0066]The reaction fluid wells 222 may be disposed within the housing 202, such as between the substrates (e.g., between the top substrate 102-2 and the base substrate 102-1 as illustrated by FIG. 1 B or otherwise contained by the base substrate 102-1 and side substrates 102-3,102-4 as illustrated by FIG.

1 D). For example and referring back to FIG. 1 B, FIG. 1 B illustrates a respective reaction fluid well 122-1 that is disposed between the top substrate 102-2 and the base substrate 102-1 . The reaction fluid wells may fluidically couple to the interconnected chamber 104, as illustrated by the reaction fluid well 122-1 fluidically coupling to the interconnected chamber 104 and the fluidic inlet 110-1. [0067] In some examples, the DMF device 100 may further include a plurality of blister packs. Referring back to FIG. 2, the reaction fluid wells 222 may couple to the plurality of blister packs which contain the reaction fluids 220. The blister packs may provide the reaction fluids 220 to the reaction fluid wells 222. In some examples, the plurality of blister packs may be disposed on the top substrate of the DMF device 200 (e.g., top substrate 102-2 of FIG. 1 B) or otherwise disposed on the housing 202 (e.g., side substrate 102-4 of FIG 1 D) and each blister pack couples to a respective reaction fluid well of the plurality 222 through a fluidic inlet of the plurality of fluidic inlets 210-1 , 210-2, 210-3, 210-4, 210-4. In some examples, the plurality of blister packs may be disposed within the reaction fluid wells 222 or within other wells on the DMF device 200 that fluidically couple to the reaction fluid wells 222. In further examples, the plurality of blister packs may include wells, which in response to being pierced, pressure within the blister pack equilibrates with atmospheric pressure such that the DMF device 200 may pull fluid from the blister pack. [0068] FIG . 1 D illustrates an example of a blister pack 116 coupled to the DMF device 100 illustrated by FIGs. 1 B-1 C, as illustrated by the top substrate 102-2 in FIG. 1 D. A blister pack, as used herein, refers to or includes a chamber containing fluid, sometimes referred to as “blister”, and a layer of breakable material coupled to the chamber. The chamber 118 may be formed of a flexible material. Breakable material, as used herein, refers to or includes material which may be pierced, torn, or otherwise broken. The breakable material 124 may include aluminum foil, plastic, or other types of materials which may be pierced and/or otherwise break. More particularly, FIG. 1 D illustrates a respective fluidic inlet 110-1 of the plurality of fluidic inlets coupled to the blister pack 116. Prior to breaking the layer of breakable material 124, reaction fluid 115 is contained within the blister pack 116 and the blister pack 116 is coupled to the fluidic inlet 110-1 of the DMF device 100. In some examples, the blister pack 116 may be coupled to the fluidic inlet 110-1 of a plurality of fluidic inlets, and the fluidic inlet 110-1 is coupled to a reaction fluid well fluidically coupled to the chamber of the DMF device 100, such as the reaction fluid well 122-1 and the interconnected chamber 104 of the DMF device 100 of FIG. 1 B.

[0069] Referring back to FIG. 1 D, a force 119 may be applied to the chamber 118 and the layer of breakable material 124 to cause the blister pack 116 to fluidically couple to the DMF device 100, such as the reaction fluid well 122-1 and/or to the interconnected chamber 104 of the DMF device 100 of FIG. 1 B. For example and referring to FIG. 1 D, the chamber 118 of the blister pack 116 is formed of a flexible material, such that a force 119 (e.g., pressing) on the flexible material causes pressure on the layer of breakable material 124 via the reaction fluid 115 filled therein and causes the layer of breakable material 124 to break. In some examples, piercing structures 123 may be located below the layer of breakable material 124 to assist with breaking the breakable material 124. In response to the break, the reaction fluid 1 15 from the chamber 118 of the blister pack 116 flows to a channel 125 that is coupled to the fluidic inlet 110-1 the DMF device 100. A piercing structure, as used herein, refers to or includes an object with a sharp point or edge. The blister pack 116 may be pierced manually by a user and/or by a piercing structure of a driving instrument that the DMF device 100 is inserted into.

[0070] Referring back to FIG. 1 B, a carrier fluid 114 may be contained between the bottom surface 109 and the top surface 107 of the interconnected chamber 104 of the DMF device 100. As noted above, the carrier fluid 114 may be used to flow the plurality of reaction fluids, as fluid droplets, through the interconnected chamber 104.

[0071 ] In some examples, the plurality of reaction fluids, as illustrated by the respective reaction fluid 115-1 , 115-2, may include aqueous fluids and the carrier fluid 114 may include an oil fluid. For example, the carrier fluid 114 may include an oil. In some examples, the carrier fluid 114 may include a silicon oil or fluorinated oil, such as FC-40 or FC-3283. Non-limiting examples of the carrier fluid 114 include FC-40, FC-43, FC-77, fluorophoroheptane (FC-84), FC- 3283, perfluoro-n-octane, perfluorodecalin, perfluorophenanthrene, perfluorohexyloctane, octofluoropropane, decafluorobutane, perfluoropentane, perfluorohexane, perfluorooctane, decafluoropentane, perfluoro(2-methyl-3- pentaone), perfluoro-15-crown-5-ether, bis-(perfluorobutyl) ethane, perfluorobutyl tetrahydrofuran, bi-perfluorohexyl ethane, perfluoro-n-hexane, perfluorooctyl bromide, perfluorotributylamine, perfluorotripentylamine, and perfluorotripropylamine, among others. In some examples, the carrier fluid 114 may include a non-fluorinated oil, such as polyphenylmehtylsiloxane, polydimethylsiloxane, hexadecane, tetradecane, octadecane, dodecane, mineral oil, isopar, or squalene. However examples are not so limited and may include other types of carrier fluids and reaction fluids that are immiscible. [0072]ln some examples, the carrier fluid 114 may be oxygenated to assist with the cell-free protein synthesis. For example, the cell-free protein synthesis reaction may use oxygen. In some examples, one of the reaction fluids may include a decomposition of peroxides which may be combined with other reaction fluids, such as the enzyme reagent, to provide oxygen for the reaction. [0073]FIG. 1 C illustrates a top view of the DMF device 100. In some examples, as shown by FIG. 1 B and 1 C, the plurality of fluidic inlets 110-1 , 110-2, 110-3, 110-4 are disposed on and through the top substrate 102-2. In some examples, as previously described, the top substrate 102-2 may be a lid with the ground electrode 106-5 disposed thereon. In some examples, the lid and ground electrode 106-5 may be transparent to allow for viewing of fluid flow and/or chemical operations within the DMF device 100.

[0074] Referring to FIG. 1 B, the circuitry 103 may be communicatively coupled to the 2D array of electrodes 106 to selectively actuate the plurality of electrodes 106 and, in response, to cause application of electrowetting forces on the plurality of reaction fluids to form fluid droplets of the plurality of reaction fluids and to drive the selective fluid flow of the fluid droplets of the reaction fluids within the interconnected chamber 104. In some examples, fluid droplets of the plurality of reaction fluids may be formed by drawing fluid from the blister pack and/or reaction fluid wells into the interconnected chamber 104.

[0075]As described above, the reaction fluids may be contained within the blister packs and/or reaction fluid wells and drawn into the interconnected chamber 104 to form the fluid droplets of the reaction fluids using the 2D array of electrodes 106. Electrowetting forces may be generated by the electrodes to split fluid packets of reaction fluids into fluid droplets of the reaction fluids, such as splitting a fluid packet 115-1 of the respective reaction fluid 115-1 , 115-2 into the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2, as illustrated by FIG. 1 B.

[0076]FIG. 1 B illustrates a respective reaction fluid well 122-1 fluidically coupled to the fluidic inlet 110-1 and is used to describe an example operation for forming a fluid droplet 115-2 of the reaction fluid 115-1 , 115-2. The reaction fluid 115-1 , 115-2 may be inserted to the DMF device 100 and forms a fluid packet 115-1 , which comprises a finite number of separate fluid droplets of the reaction fluid 115-1 , 115-2, and which may be moved together within the reaction fluid well 122-1 . Respective electrodes 106-1 , 106-2, 106-5 of the 2D array of electrodes 106 may be located in the reaction fluid well 122-1 and used to form the fluid droplet 115-2 of the respective reaction fluid 115-1 ,115-2 from the fluid packet 115-1. In some examples, the reaction fluids may be inserted to the reaction fluid wells via a pipette or other object containing a volume of the reaction fluid and via the plurality of fluidic inlets. For example, the reaction fluid 115-1 , 115-2 is inserted into the fluidic inlet 110-1 and, in response, the fluid packet 115-1 of the reaction fluid 115-1 , 115-2 forms in the reaction fluid well 122-1 . Electrowetting forces split the fluid packet 115-1 into the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2. The electrowetting forces are generated by applying an electric field via the electrodes 106, and which cause individual fluid droplets of the reaction fluid 115-1 , 115-2 to pull off from the fluid packet 115-1. The electric field causes a change in conductivity and permittivity at the interface between the reaction fluid 115-1 , 115-2 and carrier fluid 114, and produces an electric force on the interface. The electric force may cause stress on the interface, which may be referred to as a Maxwell stress, or when integrated over the area of the interface, this may be referred to as the Maxwell force. By pulling on the portion of the reaction fluid 115-1 , 115-2, while the rest of fluid is held back by other forces, such as capillary forces, the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2 is broken off from the fluid packet 115-1 of the reaction fluid 115-1 , 115-2.

[0077]The following provides a specific example of forming fluid droplets from the respective reaction fluid 115-1 , 115-2 illustrated by FIG. 1 B. The reaction fluid 115-1 , 115-2, as a fluid packet 115-1 , may be pulled into a shape that contains a neck 117 via electrowetting forces, and then pulled further by the electrowetting forces, with the neck 117 breaking off to form a fluid droplet 115-2 of the reaction fluid 115-1 , 115-2. In some examples, at least two of the electrodes of the reaction fluid well 122-1 may provide electrowetting forces on the fluid packet 115-1 of the reaction fluid 115-1 , 115-2 to form the neck 117 and break off the neck 117 to form the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2 that is smaller than the fluid packet 115-1.

[0078]0nce fluid droplets are formed, the circuitry 103 may selectively actuate the electrodes of the 2D array of electrodes 106 and the magnetic unit 108 to provide electrowetting forces and magnetic forces on fluids within or proximal to respective microfluidic paths of the interconnected chamber 104 and to draw the fluids along the respective microfluidic paths to cell-free synthesize a protein, capture the synthesized protein on a functionalized magnetic bead, and selectively trapping functionalized magnetic bead to isolate and purify the protein, as further described herein

[0079]FIGs. 1 E-1 F illustrate an example implementation of the apparatus 130 including the DMF device 100 of FIG. 1A, which is a similar implementation to FIGs. 1 B-1 C but without a top substrate 102-2. The common features and components are not repeated for ease of reference. FIG. 1 E is a cross-sectional view of the interconnected chamber 104 of the example implementation of the DMF device 100 and FIG. 1 F is a side view of the example implementation. As shown by FIG. 1 E, the housing of the DMF device 100 includes a base substrate 102-1 with side substrates 102-3, 102-4, and without a top substrate 102-2 as illustrated by FIG. 1 B. Although FIG. 1 B does not illustrate side substrates, the implementation of the apparatus 130 and/or the DMF device 100 of FIG. 1 B may include side substrates.

[0080]ln some examples, the interconnected chamber 104 includes a bottom surface 109 defined by the base substrate 102-1 and a top surface 107 defined by a carrier fluid 114 disposed within the interconnected chamber 104. The carrier fluid 114 may be contained by the base substrate 102-1 and the side substrates 102-3, 102-4. By not including a top substrate, a user may more easily view fluid operations within the interconnected chamber 104 and fabrication may be simplified. By comparison, and referring back to FIG. 1 B, including a top substrate 102-2 may allow for more integrated fluid flow and prevent contamination, fluid spill, and/or other errors.

[0081 ] In such examples, as illustrated by and referring to FIGs. 1 E-1 F, the plurality of fluidic inlets 110-1 , 110-2, 110-3, 110-4 are disposed on and through the side substrate 102-4. Similar to the implementation illustrated by FIGs. 1 B- 1 C, the electrowetting forces generated by the electrodes of the 2D array of electrodes 106, via selective actuation by the circuitry 103 may draw the reaction fluids into the interconnected chamber 104 to form the fluid droplets of the reaction fluids, as illustrated by the example fluid droplet of the reaction fluid 115, and may further drive selective flow of the fluid droplets of the reaction fluids within the interconnected chamber 104 to drive cell-free protein synthesis. Similarly, the magnetic unit 108 may be selectively actuated to trap the functionalized magnetic bead(s) and to separate the synthesized protein from other components within the plurality of reaction fluids. Although not illustrated, a reaction fluid well may be located between the fluidic inlet 110-1 and the interconnected chamber 104, similar to the reaction fluid well 122-1 illustrated by FIG. 1 B. In some examples, a blister pack may couple to the fluidic inlet 110- 1 , similar to the blister pack 116 illustrated by FIG. 1 D.

[0082]As noted above, in some examples, the 2D array of electrodes 106 may not be disposed on the base substrate 102-1 of the DMF device 100. FIG. 1 G illustrates an example implementation of the DMF device 100 of FIG. 1 A. More particularly, FIG. 1 G is a partial view of the interconnected chamber 104 of the DMF device 100 and does not illustrate all components of the DMF device 100. As illustrated by FIG. 1 G, in some examples, the electrodes 106-1 , 106-2, 106-3 of the 2D array are disposed on or within another substrate 111 which is coupable to the base substrate 102-1 . The other substrate 111 may form part of another device 127 which includes the circuitry 103-2. For example, the other device 127 may include the driving instrument that the DMF device 100 is inserted into and which couples the 2D array of electrodes 106 and the circuitry 103-2 to the DMF device 100 via circuitry 103-1 of the DMF device 100. In various examples, the DMF device 100 may be a consumable device which may be used once and then discarded. Having the 2D array of electrodes 106 and (external) circuitry 103-2 separate from and couplable to the DMF device 100 may reduce manufacturing costs. As shown by FIG. 1 G, in some examples, the circuitry 103-1 of the DMF device 100 may include an anisotropically decoupling layer which couples the 2D array of electrodes 106 and external circuitry 103-2 (e.g., a processor and/or memory) to the DMF device 100 to move fluid droplets of the reaction fluids along the respective microfluidic paths.

[0083]ln some examples, the DMF device 100 illustrated by any of FIGs. 1 A-1 G may further include a waste reservoir. For example, and as illustrated by and referring to FIG. 4D, the DMF device 200 may further a waste reservoir 440 fluidically coupled to the interconnected chamber 204. The waste reservoir 440 may be located within the housing 202 or off device, in some examples. [0084]ln some examples, the apparatus 130 as illustrated by any of FIGs. 1 A- 1 G may further include a region to collect the protein. For example, FIG. 3 further illustrates an example region 335. The region 335 as shown by FIG. 3 may include a collection well fluidically coupled to the interconnected chamber 204 (as shown by the collection well 441 of FIG. 4H), a substrate disposed within a well fluidically coupled to the interconnected chamber 204 (as shown by the well 212 and the substrate 221 of FIG. 4J), or another portion of the interconnected chamber 204 (as shown by the other portion 460 of FIG. 4I).

[0085] Referring back to FIG. 1 B, in some examples, the DMF device 100 further including a sensor 126 disposed with another portion of the interconnected chamber 104. The sensor 126 may be disposed within the interconnected chamber 104. In some examples, the sensor 126 may measure absorbance, reflectance, and/or fluorescence. For example, the sensor 126 may be used to assess or interrogate the protein, such as assaying the protein for a particular functionality, as further described herein.

[0086] In some examples, the protein may be synthesized within about one hour, purified within about 12-24 hours, and/or assessed within about 12 hours, such that a protein is synthesized, purified, and assayed in less than two days by any of the DMF devices 100 as illustrated by FIGs. 1A-1 G. In some examples, the DMF device may be capable of synthesizing and purifying, and optionally assaying, a plurality of proteins, as further described herein.

[0087]FIG. 2 illustrates an example DMF device, in accordance with examples of the present disclosure. The DMF device 200 of FIG. 2 may comprise at least some of substantially the same features and components as the DMF device 100 illustrated by any of FIGs. 1 A-1 G, as shown by the similar numbering. For example, the DMF device 200 includes a housing 202, an interconnected chamber 204, a plurality of fluidic inlet 210-1 , 210-2, 210-3, 210-4, 210-4 (herein generally referred to as “the fluidic inlets 210” for ease of reference), and a magnetic unit 208. In various examples, the DMF device 200 includes a lid and the fluidic inlets 210 are disposed in and through the lid. The common features and components are not repeated for ease of reference. [0088]As previously described, the DMF device 200 may include the reaction fluid wells 222 which fluidically couple to the fluidic inlets 210 and to the interconnected chamber 204. The reaction fluid wells 222 contain or store the reaction fluids 220. In some examples, the reaction fluid wells 222 are disposed within the housing 202. In some examples, the reaction fluid wells 222 may each be coupled to a respective blister pack (not illustrated by FIG. 2) of a plurality of blister packs, such as the blister pack 116 illustrated by FIG. 1 D. The reaction fluid wells 222 may be disposed within the housing 202 and may fluidically couple to the interconnected chamber 204. Each of the fluidic inlets 210 may be fluidically coupled to a different reaction fluid well, with each of the reaction fluid wells 222 being fluidically coupled to the interconnected chamber 204. As described above, in examples including blister packs exposed externally to the housing 202, each of the plurality of blister packs may couple to a respect one of the reaction fluid wells 222 through a respective one of the fluidic inlets 210. In other examples, the blister packs may be contained in the reaction fluid wells 222 or in other wells coupled to the reaction fluid wells 222. [0089] Reaction fluids may be inserted into the reaction fluid wells 222, for example, via pipette or other fluid source. In other examples, the reaction fluids 220 may be self-contained in the blister packs and/or reaction fluid wells 222. For example, a plurality of blister packs may disposed within or on the housing 202 and couple to a plurality of reaction fluid wells, as previously illustrated by FIG. 1 D. The blister packs may be pierced by another instrument, such as by inserting the DMF device 200 into a driving instrument containing a piercing structure. For example, and referring to FIG. 1 D, the structure may be located in the driving instrument proximal to where the DMF device 100 is disposed or inserted in, and in response to inserting the DMF device 100 into the instrument, the breakable material 124 of the blister pack 116 is pierced. In other examples, the blister packs may be pierced manually by a user using the piercing structure, such as mechanical plunger with a sharp end. Referring back to FIG 2, in other examples, the fluid flow may be caused by the electrodes and in response to the reaction fluids 220 being input to the DMF device 200, such as via a pipette, and with or without the use of blister packs. [0090]As shown by FIG. 2, a plurality of electrodes may be arranged in a 2D array of electrodes 206, which may be used to provide localized resolution of the electric field to provide fluid droplet formation, thermal zones, and selective control of fluid flow of fluid droplets of the reaction fluids. In the example illustrated by FIG. 2, the 2D array of electrodes 206 is arranged as an array that includes rows and columns of electrodes forming a rectangular shape. However, examples are not so limited and other shaped arrays may be formed that include electrodes in two dimensions. For example, the 2D array of electrodes 206 may have a variety of different arrangements and sizes and may include more or less electrodes than illustrated.

[0091 ]FIG. 3 illustrates an example apparatus including a DMF device and coupled circuitry, in accordance with examples of the present disclosure. The apparatus 330 comprises a DMF device 200, a magnetic unit 208, a 2D array of electrodes 206, and circuitry 203.

[0092]The apparatus 330 and DMF device 200 may include the apparatus 130 illustrated by FIG. 1 A and/or the DMF device 100, 200 illustrated by FIGs. 1 A- 2, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as any one of the examples DMF devices 100, 200 as described in association with any of FIGs. 1 A-2. For example, the DMF device 200 includes a housing 202 including an interconnected chamber 204 that defines a plurality of microfluidic paths, and a plurality of fluidic inlets 210 fluidically coupled to the interconnected chamber 204 to input the plurality of reaction fluids. The details of the common features and components are not repeated for ease of reference.

[0093]As described above, reaction fluids 220 may be contained in the reaction fluid wells 222 that couple to the interconnected chamber 204. The reaction fluids 220 may include or contain a functionalized magnetic bead, an enzyme reagent, and a nucleic acid template that encodes a protein. For example, the reaction fluids 220 may include a plurality of buffer fluids, with a buffer fluid of the plurality of buffer fluids containing a plurality of functionalized beads. [0094]The functionalized magnetic bead(s) may be enveloped by a functional layer and/or may be porous, and may separate molecules in the reagent fluids based on size, chemical properties, and/or combinations thereof. In some examples, the functionalized magnetic bead(s) includes a carboxylate group, a quaternary ammonium group, or a C18 tail on a surface of the bead(s).

[0095]The apparatus 330 further includes a magnetic unit 208 coupled to and disposed along the interconnected chamber 204. In some examples, the magnetic unit 208 is disposed within or on a substrate of the housing 202. In some examples, the magnetic unit 208 may form part of another device, such as a driving instrument containing the circuitry 203 that the DMF device 200 is inserted into.

[0096]As previously described, the 2D array of electrodes 206 are coupled to and disposed along the interconnected chamber 204. In some examples, the 2D array of electrodes 206 are disposed within or on a substrate of the housing 202. In other examples, the 2D array of electrodes 206 form part of another device, such as a driving instrument containing the 2D array of electrodes 206 and the circuitry 203.

[0097]The apparatus 330 further includes circuitry 203. In some examples, the circuitry 203 is coupled to or forms part of the DMF device 200, and may track and/or control operation of the 2D array of electrodes 206 and the magnetic unit 208. Such operations may comprise activation or actuation, deactivation, and other settings, such as setting to ground or floating and timings associated therewith.

[0098]The circuitry 203 may coordinate operations of the DMF device 200 including fluid flow and/or electrowetting-caused manipulation of fluid droplets of the reaction fluids 220 within the DMF device 200, such as moving, merging, and/or splitting. Such manipulation may include causing movement fluid droplets of the reaction fluids 220 along the interconnected chamber 204 within the DMF device 200 to synthesize and to purify and/or isolate a protein from other molecules in the reaction fluids. The operations of the circuitry 203 may be operated interdependently and/or in coordination with each other.

[0099]For example, the circuitry 203 is communicatively coupled to the 2D array of electrodes 206 and the magnetic unit 208 to selectively actuate electrodes of the 2D array of electrodes 206 to move fluid droplets of the plurality of reaction fluids along respective ones of the plurality of microfluidic paths within the interconnected chamber 204 and to generate a thermal zone within the interconnected chamber 204 associated with a microfluidic path of the plurality of microfluidic paths, and selectively actuate the magnetic unit 208 to move the functionalized magnetic bead toward the magnetic unit 208 and facilitate cell- free synthesis and purification of the protein. As previously described, the interconnected chamber 204 may contain a carrier fluid, and the circuitry 203 is to selectively actuate electrodes of the 2D array of electrodes 206 to form the fluid droplets of the plurality of reaction fluids as surrounded by the carrier fluid. The fluid droplets of the plurality of reaction fluids are then sequentially moved, as further illustrated by FIGs. 4A-4H.

[00100] In some examples, the apparatus 330 further includes a driving instrument 337 including the circuitry 203, the magnetic unit 208, and/or the 2D array of electrodes 206. The driving instrument 337 may receive the DMF device 200 and may arrange or place the DMF device 200 such that the 2D array of electrodes 206 and/or the magnetic unit 208 are in contact with or proximal to the interconnected chamber 204. The driving instrument 337 may include movable or switchable magnets, thermal zones (e.g., heated or cooled zones), and/or an optical sensing device or components, such as micro imaging optics and/or fluorimetery optics or a fluorescence detector.

[00101] In some examples, the DMF device 200 further includes a region 335 used to collect the purified protein, and to optionally interrogate the protein. The region 335 may include a portion of the interconnected chamber 204, such as a third portion 460 illustrated by FIG. 4I, a collection well fluidically coupled to the interconnected chamber, such as well 441 illustrated by FIG. 4H, and/or a substrate disposed within a well fluidically coupled to the interconnected chamber 204, such as the substrate 221 and well 212 illustrated by FIG. 4J. [00102] Referring to FIG. 3, the region 335 may be used to interrogate the protein, and may be referred to as “interrogation region”. The interrogation region may be located within the interconnected chamber 204 or in a well connected to the interconnected chamber 204. The protein may be moved to the region 335 via selective actuation of electrodes of the 2D array of electrodes 206. The apparatus 330, in some such examples, may further include a sensor to interrogate the protein disposed in the interrogation region. The sensor may disposed within the interconnected chamber 204, such as sensor 126 illustrated by FIG. 1 B, or may include an optical sensing device 332 which forms part of or is coupled to the driving instrument 337. The sensor may be used to assay the protein, as described above.

[00103] In some examples, the apparatus 330 further comprises an optical sensing device 332. In such examples, the region 335 may comprise a collection well or a well containing a substrate. The optical sensing device 332 may interrogate the protein and/or assay the protein. For example, the optical sensing device 332 may illuminate the region 335 using a light source 331 and collects light emitted in response to the illumination and from the region 335. The light source 331 may include a laser light or a light emitting diode (LED). Example light sources include semiconductor lasers, helium-neon lasers, carbon dioxide lasers, LEDs, incandescent lamps, and other example radiation emitting sources. The light source 331 may emit illumination light in a wavelength range between about 350 nanometer (nm) and about 1000 nm. [00104]ln some examples, the optical sensing device 332 includes a light source 331 to illuminate the region 335, and an optical detector 333 to measure the emitted light in response. An optical detector, as used herein, refers to or includes circuitry that collects light. In some examples, the optical detector 333 includes a charged coupled device (CCD) detector. The light source 331 may include a laser light, among other light sources. The optical sensing device 332 may further include a grating, filters, and/or other non-illustrated optical components, and which may be disposed between the region 335 and the optical detector 333. In some examples, additional non-illustrated optical components may be disposed between the light source 331 and the region 335 to collimate, filter, and/or focus the illumination light prior to impinging on the region 335.

[00105]Non-limiting examples of an optical sensing device 332 include a hyperspectral camera, a line scanning spectrophotometer, a fluorescence detector or fluorimeter, an ultraviolet-visible (UV-vis) spectrometer, and others. Further, the optical sensing device 332 may also include optical objectives to collect light emitted (e.g., scattered) from the region 335 and direct the emitted light to the optical detector 333. For example, a UV-vis spectrometer may be used and may operate in a V-shape, where the left side of the V-shape includes a path of illumination light provided by the light source 331 toward to the region 335 including the protein, a mirror is disposed underneath the region 335, and right side of the V-shape includes a path of light emitted from the region 335 in response to the illumination light and toward the optical detector 333. [00106]Example DMF devices and/or apparatuses may include variations from that illustrated by FIGs. 1A-3. As noted above, such variations may include, but are not limited to, the number of fluidic inlets and/or fluidic inlets, the number of electrodes, and/or arrangement of electrodes, among others.

[00107]FIGs. 4A-4J illustrate operation of an example DMF device, in accordance with examples of the present disclosure. The operation may be implemented using any of the apparatuses illustrated by FIGs 1 A-3, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as, any one of the examples DMF devices as described in association with any of FIGs. 1 A-3. The common features and components are not repeated for ease of reference.

[00108]At 441 as shown by FIG. 4A, the DMF device 200 is set to an initial state by drawing fluid droplets 442, 444, 446, 448, 449 of the plurality of reaction fluids 220 into the interconnected chamber 204 from the reaction fluid wells 222. In some examples, the initial state is caused by inserting the DMF device 200 into a driving instrument which may cause piercing of the blister packs. In other examples, a user may cause actuation of select electrodes by pressing a button on the driving instrument or otherwise instructing the circuitry 203 to begin the operation sequence. In the initial state, the fluid droplets 442, 444, 446, 448, 449 of the reaction fluids 220 are drawn into the interconnected chamber 204 and held at or near the initial position until further selective movement is activated.

[00109]The reaction fluids 220 may be used to synthesize and purify a protein via cell-free synthesis. For example, the reaction fluids 220 may include a first reaction fluid 220-1 containing the nucleic acid template that encodes the protein, a second reaction fluid 220-2 containing the enzyme reagent including a polymerase, a third reaction fluid 220-3 containing the functionalized magnetic bead, a wash buffer fluid 220-4, and an elution buffer fluid 220-5. In some examples, the first reaction fluid 220-1 contains a DNA template and the second reaction fluid 220-2 contains S30 T7. In some examples, the third reaction fluid 220-3 contains magnetic beads functionalized with nitrilotriacetic acid chelated to nickel (MagReSyn NTA-Ni). In some examples, the wash buffer fluid 220-4 is Tris-HCI, NaCI containing a first concentration of imidazone and the elution buffer fluid 220-5 is Tris-HCI, NaCI containing a second concentration of imidazone that is higher than the first concentration. However, examples are not so limited and a variety of different templates, functionalized magnetic beads, and buffer fluids may be used.

[00110]At 443 as shown by FIG. 4B, select electrodes of the 2D array of electrodes 206 are actuated to move (e.g., pull) a first fluid droplet 442 of the first reaction fluid 220-1 containing the nucleic acid template and a second fluid droplet 444 of the second reaction fluid 220-2 containing the enzyme reagent toward one other such that the first and second fluid droplets 442, 444 mix to form a first merged fluid droplet 450 in a first portion 451 of the interconnected chamber 204. Additionally at 443, respective electrodes associated with the first portion 451 of the interconnected chamber 204 are actuated to generate a thermal zone in the interconnected chamber 204 proximal to the first merged fluid droplet 450. The thermal zone may include a temperature sufficient to initiate protein expression for the protein, such as about 37 degrees C. Sufficient time is allowed for protein synthesis, such as about 1 hour or about 1 hour to about 2 hours. The remaining fluid droplets 446, 448, 449 are held at or near the initial position.

[00111 ]At 445 as shown by FIG. 4C, select electrodes of the 2D array of electrodes 206 are actuated to move (e.g., pull) a third fluid droplet 446 of the third reaction fluid 220-3 containing the functionalized magnetic beads and the first merged fluid droplet 450 toward one another such that the first merged fluid droplet 450 and the third fluid droplet 446 mix to form a second merged fluid droplet 452 containing the synthesized protein 455 and the functionalized magnetic beads. Sufficient time is allowed for synthesized protein 455 to bind to a functionalized surface of a functionalized magnetic bead. Although FIG. 4C illustrates both the first merged fluid droplet 450 and the third fluid droplet 446 being moved to another portion of the interconnected chamber 204, in some examples, the third fluid droplet 446 is brought to or near the first portion 451 of the interconnected chamber 204. The remaining fluid droplets 448, 449 are held at or near the initial position.

[00112]The synthesized protein 455 may bind to functionalized magnetic beads. In some examples, magnetic beads functionalized with NTA, such as MagReSyn NTA which are chelated to nickel, may bind to histidine tags on the protein 455. Weakly bound or unbound species may be washed away and, once complete, an elution buffer fluid containing imidazole may be added to displace the histidine-tagged protein from the functionalized magnetic bead.

[00113]As another example, magnetic beads may be functionalized with a quaternary ammonium group which enables strong anion exchange. Proteins that possess a net negative charge may bind to these resin. These proteins may be displaced through the addition of elution buffer containing salts. As a further example, magnetic beads may be functionalized with an amine group, which bind to proteins via ionic interactions.

[00114]ln some examples, the magnetic beads may be chemically functionalized with a protein, e.g., an antibody or other protein that bind to the synthesized protein. The antibody or other protein may be used to bind to a synthesized protein in a complex mixture, such as via protein tags.

[00115]At 447 as shown by FIG. 4D, the second merged fluid droplet 452 is moved toward the magnetic unit 208. In some examples, the second merged fluid droplet 452 may be moved by selective actuation of electrodes of the 2D array 206 to move the second merged fluid droplet 452 toward a second portion 456 of the interconnected chamber 204 containing or otherwise associated with the magnetic unit 208, followed by actuation of the magnetic unit 208 to draw and/or trap the functionalized magnetic beads on or near the magnetic unit 208. In some examples, a fourth fluid droplet 448 of wash buffer fluid 220-4 may be drawn from the initial position and/or formed from wash buffer fluid 220-4 contained in the respective reaction fluid well 222-4.

[00116]At 453 as shown by FIG. 4E, the fourth fluid droplet 448 of wash buffer fluid 220-4 is moved toward the magnetic unit 208 and over the second merged fluid droplet 452 via selective actuation of respective electrodes of the 2D array of electrodes 206. In some examples, the magnetic field output by the magnetic unit 208 may then be deactivated to allow for the second merged fluid droplet 452 to mix with the fourth fluid droplet 448 of wash buffer fluid 220-4 to wash the functionalized magnetic beads. In such examples, the magnetic field is again actuated to draw and/or trap the functionalized magnetic beads.

[00117]At 457 as shown by FIG. 4F, portions 454 of the second merged fluid droplet 452 that are not bound to the functionalized magnetic beads may be moved from (e.g., pulled off) the second merged fluid droplet 452 and moved toward a waste reservoir 440 coupled to the interconnected chamber 204. The waste reservoir 440 may form part of the DMF device 200 or may be separate from the DMF device 200, and/or may fluidically couple to the interconnected chamber 204 via a fluidic outlet. In some examples, to pull the portions 454 from the second merged fluid droplet 452, select electrodes of the 2D array 206 associated with a microfluidic path to the waste reservoir 440 may be actuated to draw fluids containing the portions 454 while actuating the magnetic unit 208 to attract the functionalize magnetic beads of the second merged fluid droplet 452, such that the portions 454 are flown to the waste reservoir 440 and the functionalized magnetic beads with bound proteins remain at or near the magnetic unit 208. The portions 454 may include molecules, and other components of the reaction fluid and the buffer fluid(s), e.g., waste fluids.

[00118] At 459 as shown by FIG. 4G, a fifth fluid droplet 449 of elution buffer fluid 220-5 is moved toward the magnetic unit 208 and over the second merged fluid droplet 452 via selective actuation of respective electrodes of the 2D array of electrodes 206. In some examples, the magnetic field output by the magnetic unit 208 may be deactivated to allow for the second merged fluid droplet 452 to mix with the fifth fluid droplet 449 of elution buffer fluid 220-5. The elution buffer fluid 220-5 may cause proteins bound to functionalized magnetic bead to unbind and separate from the functionalized magnetic beads, and to merge with fluid in the fifth fluid droplet 449. The wash buffer and elution buffer may be repeated multiple times to purify the protein 455.

[00119] In various examples, the fifth fluid droplet 449 containing the eluted and purified protein 455 may be moved to a region of the DMF device 200 for further analysis and/or processing. In some examples, the region includes a collection well 441 as shown by FIG. 4H. For example, at 461 as shown by FIG. 4H, the fifth fluid droplet 449 containing eluted and purified protein 455 may be moved toward a collection well 441 via selective actuation of respective electrodes of the 2D array of electrodes 206 associated with a path to the collection well 441 , and with concurrent actuation of the magnetic unit 208 to output a magnetic field and draw or trap the functionalized magnetic beads at or near the magnetic unit 208. The collection well 441 may form part of the DMF device 200 and a user may access the same to collect the protein 455 and perform additional steps off device, such as performing buffer exchange to remove imidazole or other elution buffer fluid components and/or interrogating the protein functionality by assaying the protein 455. In other examples, the protein 455 may be modified within the DMF device 200 to assay the protein 455 and then moved to the collection well 441 and optically observed by an optical sensing device, as further illustrated by FIG. 4J.

[00120] In some examples, the region may include another portion 460 of the interconnected chamber 204 of the DMF device 200 as shown by FIG. 41. For example, at 463 as shown by FIG. 41, the fifth fluid droplet 449 containing the eluted and purified protein 455 may be moved to the other portion 460 of the interconnected chamber 204 via selective actuation of respective electrodes of the 2D array of electrodes 206 associated with a path to the other portion 460, and with concurrent actuation of the magnetic unit 208 to output an magnetic field and draw or trap the functionalized magnetic beads at or near the magnetic unit 208. The other portion 460 may be associated with a transparent optical window in the top substrate of the DMF device 200 such that the other portion 460 may be optically observed by an optical sensing device, as further illustrated by FIG. 4J. In other examples, the portion 460 may include a sensor, such as the sensor 126 disposed within the interconnected chamber 204 as illustrated by FIG. 1 B.

[00121]Although not illustrated, the DMF device 200 may include an additional reaction fluid well to contain a reaction fluid containing a chemical substrate. In other examples, one of the reaction fluid wells 222 may be emptied of the respective reaction fluid and subsequently filled with the reaction fluid containing the chemical substrate. A chemical substrate, as used herein, refers to or includes a molecule or compound that reacts with a reagent, such as the protein 455, to generate a product. Referring to FIG. 4I, a fluid droplet of the reaction fluid containing the chemical substrate may be formed and then moved to the other portion 460 to merge with the fifth fluid droplet 449 containing the eluted and purified protein 455 via selective actuation of respective electrodes of the 2D array of electrodes 206 associated with a path to the other portion 460, and with concurrent actuation of the magnetic unit 208 to output an magnetic field and draw or trap the functionalized magnetic beads at or near the magnetic unit 208. In response, the other portion 460 may be interrogated to identify whether or not a product formed in response to the chemical substrate mixing with the fifth fluid droplet 449 either by an on-device sensor (e.g., sensor 126 of FIG. 1 B) or off-device sensor (e.g., optical sensing device 332 of FIG. 4J). In other examples, the third merged fluid droplet containing the fifth fluid droplet 449 and the fluid droplet of the reaction fluid containing the chemical substrate is flowed to the collection well (e.g., collection well 441 of FIG. 4H) or another well with substrate (e.g., well 212 and substrate 221 of FIG. 4H), as described below. [00122]As a non-limiting example of a chemical substrate, in some instances, the synthesized protein may be an enzyme which is a phosphatase. In such examples, a chemical substrate of 4-nitrophenyl phosphate may be used which changes color, such as to yellow, when the phosphate group is cleaved by the enzyme. A sensor or optical sensing device, such as a spectrophotometer, may be coupled to the DMF device 200 to monitor real-time changes in absorbance as the chemical substrate is modified to form the product. Other chemical substrates with different optical properties (e.g., fluorogenic) may be used depending on the detection technique used. [00123]ln some examples, the region may include an additional well 212 containing a substrate 221 of the DMF device 200 as shown by FIG. 4J. The additional well 212 may be fluidically coupled to the interconnected chamber 204, such as via a fluidic outlet 224 fluidically coupled to the interconnected chamber 204 and the well 212. A fluidic outlet refers to or includes an outlet port, e.g., an aperture, that is fluidically coupled to the interconnected chamber 204. At 465 as shown by FIG. 4J, the fifth fluid droplet 449 containing the eluted and purified protein 455 may be moved toward the well 212 via selective actuation of respective electrodes of the 2D array of electrodes 206 associated with a path to the well 212, and with concurrent actuation of the magnetic unit 208 to output an magnetic field and to draw or trap the functionalized magnetic beads at or near the magnetic unit 208. Further, at 465, the optical sensing device 332 may interrogate a test spot 213 of the substrate 221 by illuminating the test spot 213 with illumination light 481 and sensing light emitted back in response. Although one test spot 213 is illustrated, examples are not so limited and examples may include a plurality of test spots which the optical sensing device 332 may sample, and measure the response at each of the plurality of test spots. The average of the responses may be used to detect the proteins having a particular functionality, such as exhibiting the product, and which may be used to provide greater sensitivity as compared to one test spot.

[00124]As described above, a carrier fluid is contained in the interconnected chamber 204. The carrier fluid may include an oil, which may cause detection issues for the optical sensing device 332. In some such examples, the fifth fluid droplet 449 containing the eluted and purified protein 455 may be pulled out from the carrier fluid and into air. The eluted and purified protein 455 may be pulled out from the carrier fluid by selectively actuating the electrodes of the 2D array 206 to move the fifth fluid droplet 449 and leave the carrier fluid (e.g., oil phase), such that the eluted target molecule(s) enter an air phase.

[00125]Each of the above described operations may be controlled by the circuitry 203, as previously described. For example, the circuitry 203 may selectively actuate the electrodes of the 2D array of electrodes 206 and/or magnetic unit 208 to generate electric fields and/or magnetic fields. [00126JFIG. 5 illustrates an example functionalized magnetic bead and operation thereof in a DMF device, in accordance with examples of the present disclosure. For example, FIG. 5 may include a close-up view of an interconnected chamber 104, 204 of any of the DMF devices 100, 200 illustrated by FIGs. 1A-3, the common features and components not being repeated. In various examples, the operation 560 illustrated by FIG. 5 may occur via the selective movement of fluids and activations of magnetic fields, as illustrated by the operations of FIGs. 4A-4J.

[00127]Functionalized magnetic beads may be used to separate molecules in reaction fluid(s) based on size and/or chemical properties. Example chemical properties include charge, hydrophobicity, and binding affinities, among others. In various examples, the functionalized magnetic beads may have a functional layer or other composition to separate a cell-free synthesized protein from other components. In some examples, the functional layer may include a porous external surface, such that proteins smaller than pores of the porous surface may be incorporated inside the functionalized magnetic beads. In some examples, the functional layer may include functional groups or other compositions on a surface of the functionalized magnetic beads that bind to particular classes of molecules. For example, the functionalized magnetic beads may contain a surface with a carboxylate group, a quaternary ammonium group, and/or a C18 tail. The surface may be an exterior surface and/or an interior surface of the beads. In some examples, the surface is an exterior surface of the beads.

[00128]ln some examples, the functionalized magnetic beads have multiple functional layers or functionalities. For example, the functionalized magnetic beads may include a porous external surface, and a functional group or other composition on a surface, such as a carboxylate group, a quaternary ammonium group, and/or a C18 tail on an interior surface and/or exterior surface of the bead. An interior surface of a bead, as used herein, refers to or includes a surface accessible through a pore of the porous surface or otherwise not exposed to the environment with a solid or non-porous bead. An exterior surface of bead, as used herein, refers to or includes a surface exposed to the environment, and which may be exposed to molecules of any size. By including functional groups or other compositions that bind to the protein on the inside of the bead, the protein may be incorporated inside the bead and prevented and/or mitigated from escaping prematurely. As a non-limiting example, a functionalized magnetic bead may be 10 pm in diameter with 1 pm diameter pores, and proteins (e.g., peptides) of up to 400 amino acids in length may fit through the pores. However, examples are not so limited.

[00129]As shown at 562 of FIG. 5, the operation 560 may include mixing a fluid droplet containing a functionalized magnetic bead 563 with fluid droplet, such as a merged fluid droplet, containing the protein 555 and other components 565. Although FIG. 5 illustrates a functionalized magnetic bead within the fluid droplet, the fluid droplet may contain a plurality of functionalized magnetic beads in various examples.

[00130]As shown at 564, the protein 555 may bind to the functionalized magnetic bead 563. As shown at 566, a magnetic field is applied using the magnetic unit 508 to attract the functionalized magnetic bead 563. While the magnetic field is applied, a fluid droplet of wash buffer fluid is flown to and mixed with the fluid droplet to discard fluid containing unbound molecules.

[00131 ]At 568, the magnetic field is removed and the various fluid is mixed with a fluid droplet of elution buffer fluid to elute out the protein, such as the illustrated protein 555.

[00132]At 570, a magnetic field is applied using the magnetic unit 508 to attract the functionalized magnetic bead 563, while the elution buffer fluid with the protein 555 is flown to an interrogation region, such as substrate 512, for interrogation by sensor and/or an optical sensing device.

[00133]ln various examples, the DMF device may include additional number of components than illustrated, such as additional reaction fluid wells and/or additional electrodes. The additional components may be used to perform multiple purification steps, to revise the purification, and/or to perform multiplexing including synthesizing multiple different proteins.

[00134]FIGs. 6A-6B illustrate other example DMF devices, in accordance with examples of the present disclosure. FIG. 6A illustrates another example DMF device 601 which may include multiple of the same components as the DMF device 200 illustrated by FIG. 2, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as any one of the examples DMF as described in association with any of FIGs. 1 A-3. For example, the DMF device 601 includes a housing including an interconnected chamber 604 that defines a plurality of microfluidic paths, and a plurality of fluidic inlets fluidically coupled to the interconnected chamber 604 to input the plurality of reaction fluids, but with each of the regions 600-1 , 600-2, 600-3, 600-4, 600-5, 600-6, 600-7, 600-8, 600-8, 600-9 (herein generally referred to as “the regions 600” for ease of reference) corresponding to the DMF device 200, and including an additional well 612, fluidic outlet 624, and substrate 621. Accordingly, the DMF device 601 includes a scaled-up version of the DMF device 200 of FIG. 2. For example, each region 600 is coupled to a plurality of dedicated reaction fluid wells and including fluidic inlets and includes a portion of the interconnected chamber 604. Further, the 2D array of electrodes is expanded to include additional electrodes and to span the interconnected chamber 604. The details of the common features and components are not repeated for ease of reference and not all illustrated. [00135] The DMF device 601 may be used to synthesize and purify a plurality (e.g., nine) of protein variants 655-1 , 655-2, 655-3, 655-4, 655-5, 655-6, 655-7, 655-8, 655-9 (herein generally referred to as “the proteins 655” for ease of reference), such as a protein per region 600. The proteins 655 may be synthesized using a plurality of different nucleic acid templates, each input via a respective reaction fluid well associated with one of the regions 606. By scaling the DMF device 601 , N different proteins may be expressed and purified. Each electrode may be referred as a pixel and which may be dimensioned between about 50 pm by about 50 pm and about 2 by about 2 mm with a DMF device that may be up to 1000 by 1000 pixels. As an example, if using 65 pixel to express and purify a protein, a 1000 by 1000 pixel DMF device may express and purify 15,000 proteins.

[00136]Similar to that described in connection with FIG. 4I, the proteins 655 may be assayed by flowing in a chemical substrate to each region 600 and mixing with the proteins 655. The regions 600 may be observed to identify changes in absorbance or other properties indicating that the protein modified the chemical substrate to form a product.

[00137]FIG. 6B illustrates another example DMF device 600 which may include multiple of the same components as the DMF device 200 illustrated by FIG. 2, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as any one of the examples DMF as described in association with any of FIGs. 1 A-3. For example, the DMF device 600 includes a housing including an interconnected chamber 604 that defines a plurality of microfluidic paths, a plurality of reaction fluid wells 622-1 , 622-2, 622-3, 622-4, 622-5 to contain reaction fluids 620-1 , 620-2, 620-3, 620-4, 620-5, and a plurality of fluidic inlets 610-1 , 610-2, 610-3, 610-4, 610-5 fluidically coupled to the interconnected chamber 604 to input the plurality of reaction fluids, but with additional reaction fluid wells 622-6, 622-7 and additional fluidic inlets 610-6, 610-7 as compared to DMF device 200.

[00138] The additional reaction fluid wells 622-6, 622-7 may store a second buffer fluid containing second functionalized magnetic beads 620-6 and a second wash buffer fluid 620-7. The additional reaction fluids 620-6, 620-7 may be used to perform multiple different purification steps. For example, the protein may be engineered to contain a histidine tag and another protein tag, as previously described. In such examples, the protein may be purified using a histidine tag purification and after the purification may contain contaminants, such as imidazole and/or other contaminants. The DMF device 601 may be used to perform a second purification process to remove the contaminants by using the second buffer fluid 620-6 containing second functionalized magnetic beads which may be coated with an antibody or other binder that is specific to the other protein tag on the protein (e.g., FLAG tag).

[00139]The second purification may be performed similarly to that described above. For example, after separated the protein from the first functionalized magnetic beads, a sixth fluid droplet of a second buffer fluid 620-6 containing different functionalized magnetic beads may be merged with the protein 655 followed by merging with a seven fluid droplet of the second wash buffer fluid 620-7 to purify the protein from the contaminants. In some examples, the second wash buffer fluid 620-7 is Tris-HCI, NaCI without imidazole. As with before, the protein is eluted from the second functionalized magnetic beads using the elution buffer fluid 620-5. This process may be repeated multiple times to remove the concentration of the contaminants. In some examples, the protein may be released from the second functionalized magnetic beads. In some examples, the second functionalized magnetic beads using a FLAG peptide which is a 23 amino acid peptide, however examples are not so limited. [00140JFIG. 7 illustrates an example method for performing cell-free synthesis of a protein using a DMF device, in accordance with examples of the present disclosure. The method 780 may be implemented using any of the abovedescribed DMF devices and apparatuses.

[00141 ] At 782, the method 780 includes flowing a first fluid droplet of a first reaction fluid along a microfluidic path within an interconnected chamber of a DMF device via application of electrowetting forces by a 2D array of electrodes. The first reaction fluid contains a nucleic acid template that encodes a protein, and optionally a protein tag. At 784, the method 780 includes merging the first fluid droplet of the first reaction fluid with a second fluid droplet of a second reaction fluid containing an enzyme reagent to form a first merged fluid droplet. At 786, the method 780 includes generating a thermal zone in the interconnected chamber proximal to the first merged fluid droplet via actuation of an electrode of the 2D array of electrodes to drive cell-free synthesis of the protein within the first merged fluid droplet. At 788, the method 780 includes merging the first merged fluid droplet containing the protein with a third fluid droplet of buffer fluid containing a functionalized magnetic bead to form a second merged fluid droplet, wherein the protein is to bind to the functionalized magnetic bead. At 790, the method 780 includes applying a magnetic field to the second merged fluid droplet via a magnetic unit disposed along the interconnected chamber, and directing molecules in the second merged fluid droplet not bound to the functionalized magnetic bead to a waste reservoir to purify the protein. At 792, the method 780 includes flowing the purified protein along a second microfluidic path of the interconnected chamber to a portion of the DMF device. In some examples, the purified protein flowed along the second microfluidic path may be separated from the functionalized magnetic bead, e.g., prior to flowing.

[001 2] In some examples, the method 780 includes repeating cycles of turning off the magnetic field, allowing the functionalized magnetic bead to mix with additional fluid droplets containing wash buffer fluid, and moving the additional fluid droplets containing the wash buffer fluid and respective unbound molecules of the second merged fluid droplet to the waste reservoir. The method 780 may further include separating the purified protein from the functionalized magnetic bead by flowing a fourth fluid droplet of elution buffer fluid and merging the fourth fluid droplet of elution buffer fluid with the second merged fluid droplet to displace the purified protein from the functionalized magnetic bead.

[00143]ln some examples, the method 780 includes applying a second magnetic field to the functionalized magnetic bead with the purified protein displaced to trap the functionalized magnetic bead prior to flowing the purified protein to the portion of the DMF device.

[00144] In some examples, the method 780 further includes assessing the purified protein, as described above. For example, the method 780 may further include flowing a fourth (or fifth) fluid droplet containing a chemical substrate to the portion of the DMF device with the purified protein, and interrogating the portion for a product or flowing the fourth fluid droplet containing the chemical substrate and the purified protein to another portion of the DMF device and interrogating the other portion for the product using at least one of a sensor disposed in the interconnected chamber and an optical sensing device coupled to the DMF device. In other examples, the further actions may be performed off- device, as described above.

[00145]Examples are not limited to methods as described by FIG. 7. In some examples, other methods may be directed to forming or manufacturing a DMF device and/or an apparatus as described herein. An example method of manufacturing may include forming a housing defining the interconnected chamber, and to support a plurality of electrodes of a 2D array, and disposing the electrodes along the chamber. In some examples, the method may further include including positioning circuitry for support by the housing for actuating the plurality of electrodes and/or the magnetic unit.

[00146]Any of the described DMF devices may be formed of a variety of material formed in a stack. For example, a housing may formed of a plurality of different materials which are in layers, e.g., layers of substrates, in a stack. The different material layers may include a top (transparent) substrate material layer and/or a base substrate material layer, with etched or micromachined portions between that form the reaction fluid wells and the interconnected chamber, among other components. In some examples, at least one of the substrate layers may have electrodes formed thereof.

[00147] In some examples, the top (transparent) substrate material and/or the base substrate layer may have a low energy coating (e.g., a polytetrafluoroethylene (PTFE), such as Teflon™, fluorosilane, a polyamide, such as Kapton® FN, fluoroalkylsilane, 1 H,1 H,2H,2H- Perfluorodecyltriethoxysilane, trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane)) proximal to and/or in contact with the chambers, wells, reservoirs and/or channels of the DMF device and the electrodes, and/or a dielectric coating (e.g., a polyimide, such as Kapton®, Ethylene tetrafluoroethylene (ETFE), paralyne, alumina, silica, silicon nitride, aluminum nitride, aluminum oxide) proximal and/or in contact with the electrodes and/or the low energy coating. As used herein, a low energy coating includes and/or refers to a layer formed of a material having surface free energy less than 30 milliNewton/meter (mN/m). In some examples, the low energy coating may have a free energy of 20 mN/m, and/or may provide a contact angle hysteresis of less than about 10 degrees. [00148]The stack may additionally include a planarization layer with a thickness that is proportional to the electrodes, which may be formed of SU-8, paralyne, Polydimethylsiloxane (PDMS), acrylates, among other materials. For example, the planarization layer may have a thickness between the same thickness as the electrodes (e.g., in wells and/or chamber) to plus 100 percent of the thickness of the electrodes (e.g., two times the thickness of the electrodes). In some examples, the planarization layer has a thickness of between the same thickness as the electrodes and plus 10 percent of the thickness of the electrodes, or the same thickness of the electrodes and plus 50 percent of the thickness of the electrodes, among other ranges. The carrier fluid (e.g., an inert filler fluid) may be filled in the chambers, wells, reservoirs, and/or channels of the DMF device. The chambers, wells, reservoirs, and/or channels may be a height in the range of about 10 pm to about 2 mm. The various electrodes may be a length of about 40 pm to about 3 mm.

[00149] In some examples, the low energy coating is formed of PTFE. In some examples, the dielectric coating may be formed of a polyimide (e.g., Kapton®) for ease of deposition. In other examples, the dielectric coating may be formed of silicon nitride. In some examples, the planarization layer may be formed of the same material as the dielectric coating, such as a polyimide, and which may reduce the number of fabrication steps. In some specific examples, the stack may include a low energy coating formed of PTFE, a dielectric coating formed of a polyimide (e.g., Kapton®), and a planarization layer formed of the polyimide (e.g., Kapton®).

[00150]Although the above examples describe the flow of fluid within the chamber via electrowetting forces applied via the plurality of electrodes, examples are not so limited. In some examples, the control the flow of fluid within the wells and/or the interconnected chamber of any of the described DMF devices may be provided via ion emitters of the DMF device, instead of and/or by the electrodes. In some examples, a charge applicator may be brought into charging relation to a plate of the DMF device, whereby the charge applicator is to apply (e.g., deposit) charges onto the plate to cause an electric field which induces electrowetting movement of fluid within and through the DMF device. In some examples, the charge applicator is an addressable airborne charge depositing unit which may be brought into charging relation to the plate of the DMF device to deposit airborne charges onto the plate. In some examples, the charge applicator may be brought into releasable contact with, and charging relation to, the plate. The charge applicator may generate and apply the charges having a first polarity and/or an opposite second polarity, depending on whether the charge applicator is to build charges on anisotropic decoupling layer of the DMF device or is to neutralize charges. The first polarity may be positive or negative depending on the particular goals, while the second polarity is the opposite of the first polarity. Via such arrangements and in some examples, the DMF device may omit the electrodes, which would otherwise be used to cause microfluidic operations such as moving, merging, and/or splitting droplets within the DMF device. “Charges”, as used herein, refers to and/or ions (+/-) or free electrons.

[00151]Any of the above described device and/or substrates may include an anisotropic decoupling layer (e.g., 103-1 of FIG. 1 G). The anisotropic decoupling layer may decouple the working areas of the DMF device (e.g., the chamber) from electronics of the DMF device, such as the plurality of electrodes and/or the pressure regulator. For example, the anisotropic decoupling layer and the electrodes may be referred to as an anisotropic conductive layer, which facilitates migration of charges across the base substrate by providing lower resistivity across or through the base substrate and a higher lateral resistivity along the plane through which the base substrate extends. The decoupling may allow for the working areas of the of the DMF device, which contain fluids, to be inexpensive and consumable. The anisotropic decoupling layer may be formed of metal microparticles or nanoparticles aligned to form chains in one direction and encased in a polymer matrix (e.g., polymethylacrylate).

[00152JFIG. 8 illustrates an example device including non-transitory computer- readable storage medium, in accordance with examples of the present disclosure. The device 891 includes a processor 892 and memory. The memory may include a computer-readable storage medium 894 storing a set of instructions 895, 896. In various examples, the processor 892 and computer- readable storage medium 894 may be an implementation of or form part of the circuitry 103, 203 and/or part of a driving instrument, such that the device 891 is the driving instrument.

[00153]The computer-readable storage medium 894 may include Read-Only Memory (ROM), Random-Access Memory (RAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), flash memory, a solid state drive, Electrically Programmable Read Only Memory aka write once memory (EPROM), physical fuses and e-fuses, and/or discrete data register sets. In some examples, computer-readable storage medium 894 may be a non- transitory storage medium, where the term “non-transitory” does not encompass transitory propagating signals.

[00154]At 895, the processor 892 may selectively actuate electrodes of a 2D array of electrodes coupled to a DMF device to move fluid droplets of a plurality of reaction fluids to a first portion of an interconnected chamber of the DMF device, the plurality of reaction fluids including a functionalized magnetic bead, an enzyme reagent, and a nucleic acid template encoding a protein, and to generate a thermal zone within the first portion to drive synthesis of the protein from the nucleic acid template and bind the protein to the functionalized magnetic bead. At 896, the processor 892 may selectively actuate a magnetic unit coupled to a second portion of the interconnected chamber to attract the functionalized magnetic bead with the protein bound thereto.

[00155]ln some examples, the computer-readable storage medium 894 further includes instructions that, when executed, cause the processor 892 to selectively actuate electrodes of the 2D array of electrodes to move a fluid droplet of wash buffer fluid toward the second portion of the interconnected chamber to purify the protein. For example, the processor 892 may, while selectively actuating the magnetic unit, selectively actuate electrodes of the 2D array of electrodes to move components and fluids unbound to the functionalized magnetic bead to a waste reservoir fluidically coupled to the interconnected chamber. The processor 892 may selectively actuate electrodes of the 2D array of electrodes to move a fluid droplet of elution buffer fluid toward the second portion of the interconnected chamber associated with the magnetic unit to elute the protein from the functionalized magnetic bead, and while selectively actuating the magnetic unit, selectively actuate electrodes of the 2D of electrodes to move the protein to a region of the DMF device.

[00156]ln some examples, the computer-readable storage medium 894 further includes instructions that, when executed, cause the processor 892 to actuate electrodes of the 2D array of electrodes to move the protein to a region of the DMF device. The region, as previously described, may be selected from a collection well fluidically coupled to the interconnected chamber, a substrate disposed within a well fluidically coupled to the interconnected chamber, and another portion of the interconnected chamber. In some examples, the processor 892 may assess the protein for a functionality using a sensor signal received from at least one of a sensor disposed in the interconnected chamber at the region and an optical sensing device coupled to the DMF device.

[00157] In some examples, the computer-readable storage medium 894 further includes instructions that, when executed, cause the processor 892 to selectively actuate electrodes of the 2D array of electrodes to move a fluid droplet containing a chemical substrate and the protein toward another portion of the interconnected chamber to react the chemical substrate with the protein. [00158]The various ranges provided herein include the stated range and any value or sub-range within the stated range. Furthermore, when “about” is utilized to describe a value, this includes, refers to, and/or encompasses variations (up to +/- 10%) from the stated value.

[00159]Circuitry, such as the circuitry 103, 203, may include a processor and a memory as illustrated by FIG. 8. Circuitry may comprise a processor and associated memories, and optionally communication circuitry. Example circuitry includes a processor electrically coupled to, and in communication with, memory to generate control signals to direct operation of a DMF device, as well as the particular portions, components, operations, instructions, and/or methods, as described herein. Example control signals include instructions stored in memory to direct and manage microfluidic operations. The circuitry may be referred to as being programmed to perform the above-identified actions, functions, etc. In other examples, as described above, the circuitry 103, 203 may include an anisotropic conductive layer, such as the above-described anisotropic decoupling layer and a plurality of electrodes which are used to provide a plurality of microfluidic paths, which couples to electrodes of an external device. [00160] In response to or based on commands received and/or via machine readable instructions, the circuitry generates control signals as described above. The circuitry may be embodied in a general purpose computing device and/or incorporated into or associated with at least some of the example DMF devices, as well as the particular portions, components, electrodes, fluid actuators, operations, instructions, and/or methods, etc. as described herein.

[00161] Processor includes and/or refers to a processor that executes machine readable instructions contained in a memory or that includes circuitry to perform computations. Execution of the computer-readable instructions, such as those provided via memory of the circuitry, may cause the processor to perform the above-identified actions. The computer readable instructions may be loaded in a RAM for execution by the processor from their stored location in a ROM, a mass storage device, or some other persistent storage (e.g., non-transitory tangible medium or non-volatile tangible medium), as represented by memory. The computer readable instructions may include a sequence of instructions, a processor-executable machine learning model, or the like. In some examples, memory comprises a computer readable tangible medium providing non-volatile storage of the computer readable instructions executable by a processor of circuitry. In some examples, the machine readable tangible medium may be referred to as, and/or comprise at least a portion of, a computer program product. In some examples, hard wired circuitry may be used in place of or in combination with machine readable instructions to implement the functions described. For example, circuitry may be embodied as part of an applicationspecific integrated circuit (ASIC), a one field-programmable gate array (FPGA), and/or the like. The circuitry not limited to any specific combination of hardware circuitry and machine readable instructions, nor limited to any particular source for the computer readable instructions executed by the circuitry.

[00162]The circuitry may be implemented within or by a stand-alone device, such as a microprocessor. In some examples, the circuitry is partially implemented in driving instrument and partially implemented in a computing resource separate from the driving instrument but in communication therewith. For instance, the circuitry may be implemented via a server accessible via the cloud and/or other network pathways. In some examples, the circuitry is distributed or apportioned among multiple devices or resources.

[00163]Although specific examples have been illustrated and described herein, a variety of alternate and/or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.