EP4665861A1

EP4665861A1 - Use of tandem facial amphiphiles for protein expression

Info

Publication number: EP4665861A1
Application number: EP24709144.0A
Authority: EP
Inventors: Maheshwar ADIRAJ IYER; Luke M. SLOMINSKI; Alex J. SPICE; Khanh VU; Andrea Zanzotto
Original assignee: Nuclera Ltd
Current assignee: Nuclera Ltd
Priority date: 2023-02-16
Filing date: 2024-02-16
Publication date: 2025-12-24
Also published as: GB202302182D0; WO2024170915A1

Abstract

Provided herein are improved reagent compositions for cell-free protein synthesis. Particularly provided are improved aqueous droplet compositions having a tandem facial amphiphiles, and their use in methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields and reduce biofouling. The methods are applicable to cell-free protein expression on a microfluidic device having hydrophobic surfaces.

Description

USE OF TANDEM FACIAL AMPHIPHILES FOR PROTEIN EXPRESSION FIELD OF THE INVENTION The invention provides tandem facial amphiphiles for protein expression and droplet electrowetting operations. Provided herein are methods of increasing protein expression and reducing hypoxia and biofouling in aqueous droplets on a microfluidic device. Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) conditions, and methods for optimising CFPS to increase protein expression yields. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces and a non-aqueous filler fluid. BACKGROUND TO THE INVENTION Cell-free protein synthesis, also known as in-vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The CFPS environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. Thus, CFPS enables direct access and control of the translation environment which is advantageous for a number of applications including co- translational solubilisation of membrane proteins, optimisation of protein production, incorporation of non-natural amino acids, selective and site-specific labelling. Due to the open nature of the system, different expression conditions such as pH, redox potentials, temperatures, detergents and chaperones can be screened. Since there is no need to maintain cell viability, toxic proteins can be produced. A cell-free reaction usually takes a few hours, whereas in vivo protein expression may take 1 to 2 weeks. Cell-free protein synthesis (CFPS) has therefore become an important tool for molecular biologists by playing a central role in a wide variety of applications. Cell-free systems can be categorized into two main classes: cell extracts and recombinant systems. Cell extracts are highly functional but complex and undefined systems. In 2001, Shimizu et al. demonstrated that a defined cell-free system called the “PURE” system (protein synthesis using recombinant elements) could be reconstituted from purified recombinant components. The biggest advantage of CFPS is that it is the quickest way to obtain an expressed phenotype (protein) from a genotype (gene). Starting with a PCR or plasmid template, in- vitro protein synthesis and functional assays can be carried out in a few hours. Moreover, it is independent of host cells. However, cell extract based systems are known to often contain nonspecific nucleases and proteases that adversely affect protein synthesis. CFPS systems are open systems that are suitable for modification by addition of external components. To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other bioproducts adsorb and/or denature to hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Langmuir 2011, 27, 13, 8586-8594). Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O₂ serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis. Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air. However, at elevated temperatures or over prolonged periods, the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops. Hence air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature suitable for enzyme activity and the duration of the synthesis needs to be prolonged for synthesized proteins levels to be detectable. On an EWoD device, cell lysate expression leads to variable outcomes. The level of protein expression in different droplets on the same device is not consistent. The inventors herein have improved the uniformity of CFPS systems on electrowetting devices by the use of particular aqueous surfactant compositions. SUMMARY Disclosed herein are improved methods and compositions for cell-free protein synthesis. Disclosed is a method comprising the use of a tandem facial amphiphile for cell-free protein synthesis. The cell-free protein synthesis may be performed in droplets on a microfluidic device, for example a digital microfluidic device bearing a plurality of electrodes. In aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes the level of biofouling and the supply of oxygen is critical. Due to the small volume of liquid, oxygen can be consumed rapidly and hypoxic conditions develop. Many processes, including CFPS and the maturation of fluorescent proteins to generate a signal require oxygen. Disclosed herein are methods for decreasing biofouling and hypoxia in aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. Disclosed herein are methods using a tandem facial amphiphile in aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. Disclosed is a method for the synthesis of a protein in aqueous droplets on an electrowetting- on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest, a tandem facial amphiphile and a cell-free expression system including enzymes for protein synthesis. Tandem facial amphiphiles are compounds having an internal hydrophobic section and at least two hydrophilic ends, for example as described in US10316057 (incorporated herein by reference). These facial detergents are two-dimensional amphiphiles with both hydrophobic and hydrophilic faces. A tandem facial amphiphile can be a compound of a formula described herein, such as Formula I: wherein L is —(CH2)n— where n is 1-10; (C5-C8)cycloalkyl; a phenyl diradical optionally substituted by 1, 2, 3, or 4 (C1-C4)alkyl groups; —C(R^x)2—; or —CH2—C(R^x)2—CH2—; where each R^x is independently H, OH, or —CH2O-Sac; each X is independently O, S, NH, CH2, triazole, or a direct bond; each m is 0 or 1; each R¹ is independently H or (C1-C20)alkyl; and each Amph is independently a moiety of Formula A:

wherein Y is CH2 or a direct bond; each R², R³, and R⁴ is independently H, OH, or O-Sac; and each Sac is independently an oxygen-linked monosaccharide, disaccharide, or trisaccharide where the compound of Formula I has at least 4 Sac groups. The following values and variables can apply to any Formula shown herein, as applicable in the context of each formula. Tandem facial amphiphiles are commercially available from companies including Avanti polar lipids: https://avantilipids.com/product-category/detergents. Tandem facial amphiphiles include compounds in the Facade® series, including Facade®-EM, Facade®-TFA1, Facade®-TEM, Facade®-TEG, Facade®-EPC or mixtures thereof. Disclosed is the use of tandem facial amphiphiles, for example Facade surfactants such as Facade TFA1 for protein expression applications. The protein expression may be performed in droplets, optionally on an electrowetting device. The droplets and/or filler fluid may be moved during expression. The filler fluid can be moved by flow through the device. The filler fluid can be replenished when a portion, or indeed all of the filler fluid can be withdrawn from the device, thereby introducing fresh filler fluid having a higher level of oxygen than the fluid which has been withdrawn. The fluid may be withdrawn using an automated flow or under gravity. Alternatively or additionally to flowing the oil, the aqueous droplets may be moved within the device, as disclosed in application WO2021/161048. The device may contain aqueous ‘dummy’ droplets which do not contain proteins and the dummy droplets may circulated within the device thereby mixing the filler fluid. Droplets which do not contain protein can be freely circulated as no issues relating to cross-contamination or biofouling arise. Alternatively or additionally to flowing the oil and/or dummy droplets, the aqueous droplets containing proteins may be continuously circulated within the device. Application WO2021/161048 discloses the beneficial effect of droplet movement. The applicants herein have identified that circulation on path in the form of a continuous loop is beneficial over simple droplet motion. The aqueous protein droplets may be circulated in a continuous loop through the filler fluid thereby mixing the filler fluid and exposing the droplets to different areas of the device. The device may also contain aqueous ‘dummy’ droplets which do not contain proteins and the dummy droplets may circulated within the device thereby mixing the filler fluid. Droplets which do not contain protein can be freely circulated as no issues relating to cross- contamination or biofouling arise. The aqueous droplets having protein may be held stationary whilst the filler fluid moves. Alternatively the aqueous droplets may move within the device. Disclosed herein is a method of synthesizing a protein, comprising using a reaction system comprising: a. at least one template nucleic acid encoding a protein of interest; and b. a cell-free protein synthesis reagent; wherein the expression occurs in the presence of tandem facial amphiphile such as Formula I: wherein L is —(CH2)n— where n is 1-10; (C5-C8)cycloalkyl; a phenyl diradical optionally substituted by 1, 2, 3, or 4 (C1-C4)alkyl groups; —C(R^x)2—; or —CH2—C(R^x)2—CH2—; where each R^x is independently H, OH, or —CH2O-Sac; each X is independently O, S, NH, CH2, triazole, or a direct bond; each m is 0 or 1; each R¹ is independently H or (C1-C20)alkyl; and each Amph is independently a moiety of Formula A: wherein Y is CH2 or a direct bond; each R², R³, and R⁴ is independently H, OH, or O-Sac; and each Sac is independently an oxygen-linked monosaccharide, disaccharide, or trisaccharide where the compound of Formula I has at least 4 Sac groups. Disclosed herein is a method of synthesizing a protein comprising using a reaction system having: a. at least one template nucleic acid encoding a protein of interest, b. a reagent composition having enzymes for protein synthesis wherein the composition is a cell lysate which has been supplemented with oxygen and additional purified protein components. The protein synthesis reaction reagent can be a mixture of cell lysate and purified components, for example a system of purified recombinant elements i.e. protein synthesis using recombinant elements (PURE). Particularly the enzymes used for protein expression can be a mixture of cell lysates and purified enzymes. The protein synthesis reaction reagent may comprise: i. synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts and water. The reaction system may also comprise additional components that increase the yield of protein expression. The additional components may be selected from the following: cell lysates derived from naturally occurring or engineered cell lines, additional protein components, or chemical entities. The chemical entities may be one or more of a polyethylene glycol, an allolactose, an aldohexose or a thiogalactopyranoside. Where the synthesis is performed on a microfluidic device, the lysate mixture can be optimised by combining reagents on the device. For example concentrations of reagents that give optimal expression can be identified by blending components at different ratios in a large number of droplets and the expression monitored in parallel to identify optimal compositions. An aspect of the invention includes an improved system for synthesizing a protein, the system having: a. at least one template nucleic acid encoding a protein of interest; and b. a cell-free protein synthesis reagent; wherein the cell-free protein synthesis reagent contains synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10- formyl 5,6,7,8-tetrahydrofolic acid (FD), salts and water and further contains one or more additional components that improves the expression on an EWoD device. The additional components may be selected from polyethylene glycol, an allolactose, an aldohexose or a thiogalactopyranoside. The cell lysate contains a mixture of proteins and other reagents obtained from a cell without purification or separation of particular proteins. The cell lysates may be derived from mammalian cells, prokaryotic cells, yeast cells, plant cells, insect cells or protozoa. The cell lysates may be derived from human embryonic kidney cells (HEK293), chinese hamster ovary cells (CHO), HeLa, BHK21, NS0, or Sp2/0 cells. The cell lysates may be derived from Escherichia coli cells, Saccharomyces cerevisiae or Pichia pastoris cells, tobacco or wheat cells, or Leishmania tarentolae. Using only cell lysates as the reaction reagent has shown to give variable and unreliable protein expression yield. Whereas, using only purified components as the reaction reagent gives a lower protein expression yield but with more consistent results. Combining cell lysates and PURE reagents within the reaction reagent for protein expression has shown a decrease in variability and a more consistent protein expression yield. The in-vitro transcription and translation may be coupled or uncoupled. The expressed protein may be fused to a peptide tag. The tag may be used for purification and/or detection. The peptide tag may be a binding tag such as poly HIS or STREP-tag. The peptide tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example the peptide tag may be GFP₁₁ and the further polypeptide GFP_1-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherry₁₁ and the further polypeptide sfCherry_1-10. The peptide tag may be CFAST₁₁ or CFAST₁₀ and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog. For example, the GFP_1-10 polypeptide amino acid sequence could be derived from sfGFP: SEQ ID NO: 1 Alternatively, the GFP1-10 polypeptide amino acid sequence could be further mutated from the sequence above to become brighter more quickly upon complementation. The sequence may have a greater than 90 % homology to any sequence mentioned herein. The sequence may have a greater than 95 % homology to any sequence mentioned herein. SEQ ID NO: 2 The GFP1-10 polypeptide amino acid sequence could also be derived from ccGFP, having a greater than 90 or 95% homology to: SEQ ID NO: 3 (ccGFP1-11) SEQ ID NO: 4 (ccGFP_1-10) SEQ ID NO 5 (ccGFP_1-10) Nucleic acid sequence to express seq ID No 5 ccGFP1-10; SEQ ID NO: 6 SEQ ID NO 7 Nucleic acid sequence to express seq ID No 7 ccGFP1-10; SEQ ID NO: 8 The complementary GFP₁₁ peptide amino acid sequence could be the following: 1. KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9) 2. KRDHMVLHEFVTAAGITGT (SEQ ID NO: 10) 3. KRDHMVLHESVNAAGIT (SEQ ID NO: 11) 4. RDHMVLHEYVNAAGIT (SEQ ID NO: 12) 5. GDAVQIQEHAVAKYFTV (SEQ ID NO: 13) 6. GDTVQLQEHAVAKYFTV (SEQ ID NO: 14) 7. GETIQLQEHAVAKYFTE (SEQ ID NO: 15) GFP₁₁ or GFP_1-10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 – 50 amino acids. Also disclosed are nucleic acid sequences for expressing particular tags. Nucleic acid sequences include SEQ ID NO: 16 SEQ ID NO: 17 These sequences may be repeated one or more times to produce a protein having multiple GFP11 domains. For example, the sfCherry1-10 polypeptide amino acid sequence could be: SEQ ID NO: 18 The complementary sfCherry11 peptide amino acid sequence could be: SEQ ID NO: 19 sfCherry11 or sfCherry_1-10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 – 50 amino acids. For example, the NFAST polypeptide amino acid sequence could be: SEQ ID NO: 20 The complementary CFAST11 peptide amino acid sequence could be: SEQ ID NO: 21 Or the complementary CFAST10 peptide amino acid sequence could be: SEQ ID NO: 22 NFAST, CFAST11, and/or CFAST10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 – 50 amino acids. The peptide tag may also be one component of a protein that forms a detectable substrate, such as a luminescent or colorigenic substrate. The protein could include beta-galactosidase, beta-lactamase, or luciferase. The protein may be fused to multiple tags. For example the protein may be fused to multiple GFP11 peptide tags and the synthesis occurs in the presence of multiple GFP1-10 polypeptides. For example the protein may be fused to multiple sfCherry11 peptide tags and the synthesis occurs in the presence of multiple sfCherry_1-10 polypeptides. The protein of interest may be fused to one or more sfCherry₁₁ peptide tags and one or more GFP₁₁ peptide tags and the synthesis occurs in the presence of one or more GFP_1-10 polypeptides and one or more sfCherry_1-10 polypeptides. Any protein of interest may be synthesised. The protein may be an enzyme, for example a terminal deoxynucleotidyl transferase (TdT) enzyme or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species. For example the protein being synthesised may be a membrane protein. Membrane proteins are the molecular gatekeepers of the cell, essential for maintaining its structural integrity, regulating molecular transport, and orchestrating communication with the external environment. These proteins are typically embedded within the lipid bilayer that forms the cell membrane. Through their diverse structures and functions, membrane proteins play pivotal roles in a myriad of cellular processes, including signal transduction, cell recognition, and adhesion. Understanding the intricate architecture and functionality of membrane proteins is crucial for unraveling the complexities of cellular biology and holds significant implications for fields ranging from medicine to biotechnology. Tandem facial amphiphiles are known to help stabilise membrane proteins. The inventors herein have appreciated that such compounds also increase the yields and ability to isolate such proteins during cell-free protein synthesis, particularly on microfluidic devices such as electrowetting devices. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the hypoxia of half-half array SBF081 (0.02% Facade TFA1) and SBF023 (0.05% Pluronic F127). The hypoxia signal appearing in the centre of the array is reduced for the droplets having the Facade. Figure 2 shows the oxygen depletion of the half-half array. The hypoxia signal appearing in the centre of the array is reduced for the droplets having the Facade. Figure 3 shows performance of 0.02% Facade TFA-1 (SBF081) and 0.05% Pluronic F127 (SBF023) side-by-side using 144-droplet array running the standard LS70/LEC assay. Figure 3 shows the incubation at 24 hours. The left-hand side of the panel shows the droplets of SBF081. The right-hand side of the panel shows the droplets of SBF023. The fluorescent intensity of SBF081 is uniform across the droplets, which are brighter than the standard 1mg/ml. The black hole is seen here in the right-hand side of the device where the SBF023 located. Figure 4 shows the drained device from Figure 3. Residual protein biofouling is greater in the half with F127 and is largely absent in the droplets having Facade. Figure 5 shows hypoxia levels and protein expression comparing SBF081 (0.02% Facade TFA1) and SBF023 (0.05% Pluronic F127). Facade TFA1 surfactant has an impact on O₂ consumption or diffusion in a way that reduced the black hole on a device. The area of hypoxia intensity spread 8 droplets horizontally by 8 droplets vertically in SBF023, whereas it only spread 6 droplets horizontally by 6 droplets vertically in SBF081. Standing out, LUPA with TFA1 shows no black hole under GFP and the least hypoxia showed, which is only 3 droplets x 2 droplets. Figure 6 in-tube fluorescence comparing surfactant compositions. Figure 7 graphical representation of expression yield comparing surfactant compositions. Figure 8 shows droplets becoming immobilised (pinning) during an expression reaction of two proteins from several timepoints in an expression incubation and the beginning of post-split, illustrating that the droplet pinning without a tandem facial amphiphile started to occur within 1 hour of incubation across the array. Pinning of droplets was observed throughout from expression to incubation, when they were unable to split. However, both constructs did not experience any droplet handling issues in the presence of the tandem facial amphiphile (Facade TFA-1). At the end of complementation, the split without the Facade failed; and droplets remained immobile for the rest of the droplet handling process. Constructs having Facade successfully split into smaller droplets. Columns shown A = Protein 1 in standard expression conditions (No Facade). B = Protein 1 plus Facade. C = Protein 2 in standard expression conditions. D = Protein 2 plus Facade. Columns A and C have failed to split into smaller droplets. Columns B and D behave as expected. Figure 9 shows fluorescent of the device after complete draining of the liquids. The areas showing fluorescent signal are deposited protein. The presence of Facade largely eliminates the protein fouling, which is only visible in columns where the Facade is absent. DETAILED DESCRIPTION OF THE INVENTION Disclosed is a method for decreasing hypoxia and biofouling in aqueous droplets in an aqueous immiscible filler fluid on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising improved surfactant compositions. Disclosed herein are methods using one or more tandem facial amphiphile(s) in aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. The droplets may be in an aqueous immiscible filler fluid or a humidified air filled device. Disclosed is a method for the synthesis of a protein in aqueous droplets on an electrowetting- on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest, a tandem facial amphiphile and a cell-free expression system including enzymes for protein synthesis. Disclosed is a method for the synthesis of a protein in aqueous droplets on an electrowetting- on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest and a cell-free expression reagents including enzymes for protein synthesis and moving the filler fluid in order to replenish oxygen to the aqueous droplets. Disclosed herein is a method of synthesizing a protein, comprising using a reaction system comprising: a. at least one template nucleic acid encoding a protein of interest; and b. a protein synthesis reaction reagent; wherein, the oxygen present in b. has been increased to increase the yield of protein expression. Disclosed is a method of synthesising a protein in a digital microfluidic device. The droplets having the components required for cell-free protein synthesis (CFPS), otherwise known as in-vitro protein synthesis, can be manipulated by electrokinesis in order to effect and improve protein expression. Tandem facial amphiphiles are compounds having an internal hydrophobic section and at least two hydrophilic ends, as described in US10316057 (incorporated herein by reference). The ends are typically formed of hydrophilic sugars. A tandem facial amphiphile can be a compound of a formula described herein, such as Formula I: wherein L is —(CH2)n— where n is 1-10; (C5-C8)cycloalkyl; a phenyl diradical optionally substituted by 1, 2, 3, or 4 (C1-C4)alkyl groups; —C(R^x)2—; or —CH2—C(R^x)2—CH2—; where each R^x is independently H, OH, or —CH2O-Sac; each X is independently O, S, NH, CH2, triazole, or a direct bond; each m is 0 or 1; each R¹ is independently H or (C1-C20)alkyl; and each Amph is independently a moiety of Formula A: wherein Y is CH2 or a direct bond; each R², R³, and R⁴ is independently H, OH, or O-Sac; and each Sac is independently an oxygen-linked monosaccharide, disaccharide, or trisaccharide where the compound of Formula I has at least 4 Sac groups. The following values and variables can apply to any Formula shown herein, as applicable in the context of each formula. Tandem facial amphiphiles are commercially available from companies including Avanti polar lipids: https://avantilipids.com/product-category/detergents. In some embodiments, L can be —(CH₂)_n—. The variable n can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, L can be a cycloalkyl diradical, such as a disubstituted cyclopentane, disubstituted cyclohexane, disubstituted cycloheptane, or disubstituted cyclooctane. In other embodiments, L can be a phenyl diradical. The phenyl diradical can be a 1,2-, 1,3-, or 1,4-diradical. In some embodiments, the phenyl can be substituted, such as with 1, 2, 3, or 4 (C₁-C₄)alkyl groups, e.g., a p-xylyl group. In some embodiments, n is not 1, n is not 1 or 2, n is not 1-3, or n is not 1-4. In certain embodiments, n is at least 3, 4, 5, or 6 when one or both X groups is CH₂. In some embodiments, n is not 3. In some embodiments, n is 1-10 and R¹ is (C₅-C₂₀)alkyl. In some embodiments, R⁴ is H. In various embodiments, m=0. In certain embodiments, one X is not CH₂. In other embodiments, both X groups are not CH₂. In some embodiments, L can be a —C(R^X)₂—; or —CH₂—C(R^X)₂—CH₂— linking group. Each R^x can independently be H, OH, or —CH₂O-Sac. When R^x is —CH₂O-Sac, it can be represented by —CH₂—R² where R² is O-Sac. In some embodiments, each m is 0. In other embodiments, m is 1. When m is 0, Y is typically CH2, although it can also be a direct bond. When m is 1, Y is typically a direct bond, although it can also be CH2. In some embodiments, each R¹ is H. When R¹ is H, the compounds typically make good hydrogel compositions with water. In other embodiments, each R¹ is (C1-C20)alkyl. When R¹ is (C1-C20)alkyl, the alkyl can be straight chain, branched, or optionally substituted. Examples of (C1-C20)alkyl groups include the groups recited in the definition of alkyl. In some embodiments, each X is O. In other embodiments, each X is S. In other embodiments, each X is NH. In yet other embodiments, each X is CH2. In further embodiments, X can be a triazole diradical or a direct bond. Each R², R³, and R⁴ can independently be H, OH, or O-Sac, such that each amphiphile includes at least 4 Sac moieties. Each Sac is independently an oxygen-linked monosaccharide, disaccharide, or trisaccharide. Specific examples of Sac groups are recited in the definition of the term saccharide. In one specific embodiment, the compound has 4 Sac groups. In another specific embodiment, the compound has 6 Sac groups. In another specific embodiment, the compound has 8 Sac groups. In one specific embodiment, each m is 1 and Y is a direct bond. In one specific embodiment, each R¹ is (C₁-C₂₀)alkyl. In one specific embodiment, each X is a direct bond. In one specific embodiment, L is —(CH₂)_n— where n is 1-6. In one specific embodiment, Y is a direct bond. In one specific embodiment, R⁴ is H. In one specific embodiment, R² and R³ are O-Sac and each Sac is a disaccharide. In one specific embodiment, R² and R³ are O-Sac and each Sac is a maltosyl group. The compound of Formula I can be a compound of any one of Formulas II-XIV.

In an embodiment, the compound is a compound of Formula IV where each R¹ is (C1-C8)alkyl. In other embodiments, each R¹ is CH3; CH2CH3; CH2(CH3)2; (CH2)3CH3; or (CH2)4CH3. In one embodiment, the compound is a compound of Formula VII (i.e., Formula I where m is 0 and Y is CH2), where each X is independently O, S, NH, CH2, or triazole; and n is −1, 0, 1, 2, 3, 4, 5, or 6. In one embodiment, the compound is a compound of Formula VIII where each X is S, O, CH2, or a triazole diradical. In another embodiment, the compound is a compound of Formula IX where each R² is independently a monosaccharide, disaccharide, trisaccharide. In one embodiment, each Sac is a monosaccharide. In another specific embodiment, each Sac is a monosaccharide or a disaccharide. In another specific embodiment, each Sac is a disaccharide. In another specific embodiment, each Sac is a trisaccharide. In another embodiment, the compound is a compound of Formula XII where each R² is independently an oxygen linked monosaccharide, disaccharide, trisaccharide, for example glucose, maltose, or raffinose. In some embodiments, R², R³, and/or R⁴ can be —O-glucosyl, —O-maltosyl, —O-galactosyl, and the like. In certain embodiments, one or more of R², R³, and/or R⁴ can exclude —O- maltosyl groups.

Tandem facial amphiphiles are commercially available from companies including Avanti polar lipids: https://avantilipids.com/product-category/detergents. Particular examples of tandem facial amphiphiles include compounds in the Facade® series, including Facade®-EM, Facade®-TFA1, Facade®-TEM, Facade®-TEG, Facade®-EPC or mixtures thereof: Facade®-EM: Two Dimensional Amphiphiles: https://avantilipids.com/product/850522 3α-hydroxy-7α,12α-di-((O-ß-D-maltosyl)-2-hydroxyethoxy)-cholane

Facade®-TFA1 Detergent: https://avantilipids.com/product/850526 Facade®-TEM: https://avantilipids.com/product/850537 3α,7α,12α-tri-((O-ß-D-maltopyranosyl)ethyloxy)-cholane

Facade®-TEG: 3α,7α,12α-tri-((O-β-D-glucopyranosyl)ethyloxy)-cholane Facade®-EPC: https://avantilipids.com/product/850539 3 ^-hydroxy-7 ^,12 ^-di-(((2-(trimethylamino)ethyl)phosphoryl)ethyloxy)-cholane The tandem facial compounds may be used at an amount of 0.01 to 0.1 % by volume. The compounds may be used at 0.02 % by volume. Protein expression is dependent on the conditions and reagents used for expression. The best expression system for a given protein of interest is not predictable, and may require screening of a large number of similar conditions in order to identify the optimal expression system. The use of EWoD devices can screen large numbers of closely related conditions in parallel in droplets on the device. The droplets can be blended on the device in order to prepare the reagents. For example a single cell lysate or reconstituted protein mix can be supplemented with a variety of additional components at a selection of concentrations. For example a salt screen, buffer screen or pH screen can be performed across a range of conditions and at variable concentrations. Disclosed herein is a method for the synthesis of a protein on an electrowetting-on-dielectric (EWoD) device, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest, blending droplets to form a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest. Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A/C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path. As an alternative to microfluidic channel systems, droplets can also be generated and manipulated on planar surfaces using digital microfluidics (DMF). In contrast to channel based microfluidics, DMF utilizes alternating polarity voltage signals on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation. Cell-free protein synthesis, also known as in-vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in- vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol.2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol.2014; 1118: 275–284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268). Protein expression using cell lysates typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O₂ serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis. Levels of oxygen can be increased by agitating/mixing the filler basefluid. Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and a cell-free system having components for protein expression in an oil-filled environment, and moving said oil. The components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device. Mixing of the filler fluid can be performed using dummy droplets. The dummy droplets typically do not contain proteins or other biomolecules. The dummy droplets may be larger in size than the droplets expressing proteins. The dummy droplets can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed in adjacent droplets. The droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen to be supplied to the adjacent droplet and dispersed throughout the droplets on the device. The act of moving improves the level of protein expression over a droplet which remains static. Alternatively or additionally the protein droplets can be moved within the device. The dummy and/or protein droplets can be circulated in a continuous loop. The movement of the droplets in a path through the oil increases the oxygen available to the droplets. In order to further increase the mixing of the oil, the aspect ratio of the droplets can be changed in order to increase the exposed surface area displacing the filler fluid. Thus rather than moving a square droplet, the droplet can be made rectangular and moved with the longer side as leading edge in order to increase the filler fluid displacement. The droplets can be moved using any means of electrokinesis. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors. The filler liquid may be a hydrophobic or non-ionic liquid. For example the filler liquid may be decane or dodecane. The filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DMPS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85. The non aqueous filler fluid, or oil, in the device can be any water immiscible liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil or a blend thereof. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an air-filled device where droplets containing cell- free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C). A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be located near or merged with the droplets during the protein expression. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air. The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis. The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with additive droplets for screening. The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery. In order to optimise expression, the expression system can be supplemented with additional components, including purified enzymes. The additional components may include salts, co- factors, buffers, surfactants, chaperones or additional protein components. The additional protein components may be selected from for example chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases. The expression composition may be assembled on the device from mixing a variety of droplets in order to screen a variety of compositions in parallel. By way of example, the screening reagents may include, ■ Chaperone mix (e.g., PUREfrex GroE mix) ■ Kinase 1 (e.g., NEB CK2) ■ Kinase 2 (e.g., NEB PKA) ■ Protease 1 (e.g., NEB TEV) ■ Protease 2 (e.g., Merck HRV 3C) ■ Acetyl transferases ■ Common metal ions cocktail ■ Common co-factors cocktail The compositions can be blended by the user and the level of expression of the protein of interest monitored in each of the blended conditions. Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in-vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet. The cell-free protein being expressed may be a membrane protein. Membrane proteins encompass a wide range of functional and structural diversity, each serving specific roles crucial for cell function. Examples of different types of membrane proteins include: Ion Channels: These proteins form pores in the cell membrane, allowing the selective passage of ions such as sodium, potassium, calcium, and chloride across the membrane. Examples include voltage-gated ion channels, ligand-gated ion channels, and mechanosensitive ion channels. Transporters: Membrane transporters facilitate the movement of molecules, such as sugars, amino acids, and ions, across the membrane against their concentration gradient. Examples include glucose transporters (GLUT proteins) and ATP-binding cassette (ABC) transporters. Receptors: Membrane receptors are proteins that bind to specific signaling molecules, such as hormones, neurotransmitters, or growth factors, initiating a cellular response. Examples include G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and ligand- gated ion channels. Enzymes: Some membrane proteins catalyze biochemical reactions at the cell membrane or participate in the synthesis or degradation of molecules. Examples include adenylyl cyclase, which produces cyclic AMP (cAMP), and phospholipase C, which cleaves phospholipids to generate second messengers like inositol trisphosphate (IP3) and diacylglycerol (DAG). Structural Proteins: Membrane proteins can also contribute to the structural integrity of the cell membrane and its associated structures. Examples include integrins, which mediate cell adhesion to the extracellular matrix, and cadherins, which facilitate cell-cell adhesion. Anchoring Proteins: These proteins tether the intracellular cytoskeleton to the cell membrane, providing structural support and facilitating signal transduction. Examples include spectrin and ankyrin, which link integral membrane proteins to the cytoskeleton. Cell Surface Markers: Membrane proteins can serve as identifiers for the cell, facilitating cell recognition, adhesion, and immune responses. Examples include major histocompatibility complex (MHC) proteins and blood group antigens. The expression system may be used for expressing membrane proteins having one or more solubility tags. Integral membrane proteins (IMPs) account for nearly one third of all open reading frames in sequenced genomes and play vital roles in all cells including intra- and intercellular communication and molecular transport. Given their centrality in diverse cellular functions, IMPs have enormous significance in disease. However, understanding of this important class of proteins is hampered in part by a lack of generally applicable methods for overexpression and purification, two critical steps that typically precede functional and structural analysis. Most IMPs are naturally of low abundance and must be overproduced using recombinant systems. However, the yields of chemically and conformationally homogenous, active protein following overexpression in bacteria, yeast, insect cells or cell- free systems are often still too low to support functional and/or structural characterization, and can be further confounded by aggregation and precipitation issues. This limitation can sometimes be overcome using protein engineering whereby fusion partners are used to increase expression and promote membrane integration. Alternatively, mutations can be introduced to the IMP itself that enhance its stability or even render it water soluble. However, these approaches are largely trial and error, and the identification of suitable fusion partners or stabilizing mutations is neither trivial nor generalizable. Even when appropriate yields can be obtained, the hydrophobic nature of IMPs requires their solubilization in an active form, which may be achieved through the use of detergents that provide a lipophilic niche inside a detergent micelle. Because IMPs interact uniquely with each detergent, identifying the best detergents often involves lengthy and costly trials. A number of detergent-like amphiphiles have been developed that stabilize IMPs in solution including protein-based nanodiscs, peptide-based detergents, Styrene maleic-acid lipid particles (SMALPs) etc, and while these have helped to increase knowledge of IMPs, each type of amphiphile has its own limitations, and no universal reagent has been developed for wide use with structurally diverse IMPs. Methods described herein allow screening of one or more tandem facial amphiphiles to help stabilise expressed membrane proteins. Expression using tandem facial amphiphiles may be used to solubilize not only membrane proteins but also intrinsically disordered proteins or any proteins that readily unfold to expose their hydrophobic core causing aggregation. The solubility tag or decoy/shield proteins may cover up hydrophobic regions that cause soluble proteins to aggregate. The protein may be stabilized by attachment to multiple solubility tags, for example tags at both the C and N sides of the trans-membrane domain. The protein may include an amphipathic shield domain protein moiety which can act as a solubility tag; an integral membrane protein moiety; and a water soluble expression decoy protein moiety. The amphipathic shield protein moiety may be coupled to the integral membrane protein moiety's C-terminal domain and the water soluble expression decoy protein moiety coupled to the integral membrane protein moiety's N-terminal domain. The amphipathic shield protein moiety may be coupled to the integral membrane protein moiety's N-terminal domain and the water soluble expression decoy protein moiety coupled to the integral membrane protein moiety's C-terminal domain. Thus the hydrophobic protein is provided with hydrophilic solubility tags at both the N and C terminus in the form of shield and decoy proteins such as lipoproteins, for example apoliproteins such as APoE. The proteins may be membrane proteins or other proteins having intrinsically disordered regions or any proteins that readily unfold to expose their hydrophobic core causing aggregation. The proteins may have multiple solubility tags attached to ensure the membrane or hydrophobic protein is soluble in the absence of a membrane. Preparation of stabilised membrane proteins in described in US10,961,286, incorporated herein by reference in its entirety. As used herein, the term “integral membrane protein” (IMP) includes a type of transmembrane protein held in the bilayer of a cellular membrane by lipid groups with tight binding to other proteins. The IMPs of the present invention play vital roles in all cells including intra- and intercellular communication and molecular transport. The IMPs of the present invention are uniquely stable and water soluble following extraction from their native environment (e.g., a cellular membrane) without the use of detergents and/or detergent-like amphiphiles, overproduction using recombinant systems, protein engineering, and/or mutations to the IMP itself, thereby allowing for improved functional and structural studies of IMPs as well as in-vitro reconstitution of enzymatic activity or in-vitro reconstitution of a biological pathway involving water soluble IMP enzymes and engineering of biological/metabolic pathways directly in living cells involving the water soluble IMPs. The IMPs of the present invention may be selected from the group consisting of bitopic α- helical IMPs, polytopic α-helical IMPs, IMPs with multiple helices, and polytopic β-barrel IMPs. The IMPs of the present invention may be classified structurally as β-barrel or α-helical bundles. β-barrels may be expressed as inclusion bodies, purified and refolded for structural studies, whereas α-helical bundles are less likely to produce soluble active forms after refolding. In one embodiment, the bitopic α-helical IMP is human cytochrome b5 (cyt b₅). Cyt b₅ is a 134- residue bitopic membrane protein consisting of six α-helices and five β-strands folded into three distinct domains: (i) an N-terminal haeme-containing soluble domain; (ii) a C-terminal membrane anchor; and (iii) a linker or hinge region that connects the two domains. Native cyt b5 stimulates the 17,20-lyase activity of cytochrome P450c17 (17α-hydroxylase/17,20-lyase; CYP17A0). In particular, a molar equivalent of cyt b5 increases the rate of the 17,20-lyase reaction 10-fold, via an allosteric mechanism that does not require electron transfer. Given that the C-terminal transmembrane helix of cyt b5 is required to stimulate the 17,20-lyase activity of human CYP17A0, the ApoAI* shield may, in one embodiment, be sufficiently flexible to allow the protein-protein interactions that are necessary to promote proper function. In another embodiment, the polytopic α-helical IMP is selected from the group consisting of Homo sapiens hydroxy steroid dehydrogenase (HSD17β3), H. sapiens glutamate receptor A2 (GluA2), E. coli DsbB (DsbB), H. sapiens Claudin1 (CLDN1), H. sapiens Claudin3 (CLDN3), H. sapiens sapiens steroid 5a-reductase type 1 (S5αR1), H. sapiens sapiens steroid 5a-reductase type 2 (S5αR2), and Halobacterium sp. NRC-1 bacteriorhodopsin (bR). In one embodiment, a small (110 amino acids) polytopic α-helical IMP from E. coli named ethidium multidrug resistance protein E (EmrE), comprised of four transmembrane α-helices having 18-22 residues per helix with very short extramembrane loops, may be used. EmrE as described herein is the archetypical member of the small multidrug resistance protein family in bacteria and confers host resistance to a wide assortment of toxic quaternary cation compounds by secondary active efflux. In another embodiment, the polytopic β-barrel IMP is selected from the group consisting of E. coli OmpX (OmpX) and Rattus norvegicus voltage-dependent anion channel 1 (VDAC1). In another embodiment, the IMPs with multiple helices may further include, for example, polytopic β-barrel membrane proteins such as outer membrane proteins including, for example, OmpX, OmpX^a, OmpA, OmpA^a, PagP^a, NspA, OmpT, OpcA, NalP, OmpLA, TolC, FadL, OmpF, PhoE, Porin, OmpK36, Omp32, MspA, LamB, Maltoporin, ScrY, BtuB, FhuA, FepA, and FecA. See Tamm et al., “Folding and Assembly of β-barrel Membrane Proteins,” Biochimica et Biophysica Acta 1666:250-263 (2004), which is hereby incorporated by reference in its entirety. Non-constitutive β-barrel membrane proteins include, but are not limited to, α-Hemolysin and LukF. See Tamm et al., “Folding and Assembly of β-barrel Membrane Proteins,” Biochimica et Biophysica Acta 1666:250-263 (2004), which is hereby incorporated by reference in its entirety. In yet another embodiment, the IMP is selected from the group consisting of G protein-coupled receptors (GPCR) and olfactory receptors. GPCRs can include the Class A (Rhodopsin-like) GPCRs, which bind amines, peptides, hormone proteins, rhodopsin, olfactory prostanoid, nucleotide-like compounds, cannabinoids, platelet activating factor, gonadotropin-releasing hormone, thyrotropin-releasing hormone and secretagogue, melatonin and lysosphingolipid and LPA. GPCRs with amine ligands can include, without limitation, acetylcholine or muscarinic, adrenoceptors, dopamine, histamine, serotonin or octopamine receptors); peptide ligands include but are not limited to angiotensin, bombesin, bradykinin, anaphylatoxin, Fmet- leu-phe, interleukin-8, chemokine, cholecystokinin, endothelin, melanocortin, neuropeptide Y, neurotensin, opioid, somatostatin, tachykinin, thrombin vasopressin-like, galanin, proteinase activated, orexin and neuropeptide FF, adrenomedullin (G10D), GPR37/endothelin B-like, chemokine receptor-like and neuromedin U. As used herein, the term “amphipathic shield domain protein” includes any protein that displays both hydrophilic and hydrophobic surfaces and is often associated with lipids as membrane anchors or involved in their transport as soluble particles. The amphipathic shield domain protein, in one embodiment, serves as a molecular shield to sequester large lipophilic surfaces of the IMP from water. Apolipoproteins are proteins that bind lipids (oil-soluble substances such as fats, cholesterol and fat soluble vitamins) to form lipoproteins. They transport lipids in blood, cerebrospinal fluid and lymph. The lipid components of lipoproteins are insoluble in water. However, because of their detergent-like (amphipathic) properties, apolipoproteins and other amphipathic molecules (such as phospholipids) can surround the lipids, creating a lipoprotein particle that is itself water-soluble, In various embodiments, the amphipathic shield domain protein may be selected from the group consisting of Apolipoprotein A (Apo-AI, Apo-A2, Apo-A4, and Apo-A5), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein D (ApoD), apolipoprotein E (ApoE), apolipoprotein F (ApoF), apolipoprotein L (ApoL), apolipoprotein M (ApoM), apolipoprotein M (ApoM) and a peptide self-assembly mimic (PSAM). In particular, the amphipathic shield domain protein may be apolipoprotein A0 (ApoAI). As used herein, ApoAI avidly binds phospholipid molecules and organizes them into soluble bilayer structures or discs that readily accept cholesterol. ApoAI contains a globular amino-terminal (N-terminal) domain (residues 1-43) and a lipid-binding carboxyl-terminal (C-terminal) domain (residues 44-243). In one embodiment, the ApoAI may be truncated (ApoAI*). Truncated variants of ApoA0 include, but are not limited to, human ApoAI lacking its 43-residue globular N-terminal domain. As used herein, ApoA0 exhibits remarkable structural flexibility, and may adopt a molten globular-like state for lipid-free ApoAI under conditions that may allow it to adapt to the significant geometry changes of the lipids with which it interacts. The present invention designs chimeras in which, for example, ApoAI* may be genetically fused to the C terminus of an IMP target. Expression of these chimeras in the cytoplasm of Escherichia coli may yield appreciable amounts of globular, water-soluble IMPs that are stabilized in a hydrophobic environment and retain structurally relevant conformations. The approach provides, inter alia, a facile method for efficiently solubilizing structurally diverse IMPs, for example in both bacteria and human cells, as a prelude to functional and structural studies, all without the need for detergents or lipid reconstitutions. In one embodiment, a plasmid may be used which encodes a chimeric protein in which ApoAI is fused to the C-terminus of EmrE. In another embodiment, the amphipathic shield domain protein is a peptide self-assembly mimic (PSAM). The shield domain may be made of multiple proteins with optional linkers. The shield may be multiple proteins selected from apolipoprotein A (ApoA), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein D (ApoD), apolipoprotein E (ApoE), apolipoprotein H (ApoH), and a peptide self-assembly mimic (PSAM). The solubility tag may take the form of a water soluble expression decoy protein. As used herein, the term “water soluble expression decoy protein” includes any protein which serves to direct an IMP into cellular cytoplasm. The water soluble expression decoy protein may assist in “tricking” a hydrophobic IMP into thinking that it is not hydrophobic. The desired water soluble decoy protein for a particular IMP can be identified by the methods described herein by producing a variety of nucleic acid sequences expressing a shield domain protein-IMP- variety of decoy conjugates and seeing which nucleic acid construct best expresses soluble and detectable protein, thereby identifying a preferred decoy conjugate. The decoy can be attached to the C or N terminus. Disclosed is a method wherein the nucleic acid encodes a tripartite fusion protein, said nucleic acid molecule comprising: a first nucleic acid moiety encoding one or more amphipathic shield domain protein(s) selected from the group consisting of apolipoprotein A (ApoA), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein D (ApoD), apolipoprotein E (ApoE), apolipoprotein H (ApoH), and a peptide self-assembly mimic (PSAM); a second nucleic acid moiety encoding an integral membrane protein; and a third nucleic acid moiety encoding one or more solubility tag(s) in the form of a water soluble expression decoy protein. The a first nucleic acid moiety encoding an amphipathic shield domain protein and the a second nucleic acid moiety encoding an integral membrane or hydrophobic protein may be located between regions A0 and B0, and become attached to a variety of solubility tags/decoy proteins using the methods described herein. Disclosed is a method wherein the nucleic acid encodes a tripartite fusion protein, said nucleic acid molecule comprising: a first nucleic acid moiety encoding an amphipathic shield domain protein selected from the group consisting of apolipoprotein A (ApoA), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein D (ApoD), apolipoprotein E (ApoE), apolipoprotein H (ApoH), and a peptide self-assembly mimic (PSAM); a second nucleic acid moiety encoding an integral membrane protein; and a third nucleic acid moiety encoding a solubility tag in the form of a water soluble expression decoy protein, wherein said first nucleic acid moiety is coupled to said second nucleic acid moiety's 3′ end and said third nucleic acid moiety is coupled to said second nucleic acid moiety's 5′ end, said coupling being direct or indirect. The shield and/or decoy proteins may be connected to the membrane protein via a cleavable linker such as a sequence cleavable using a protease. The protease may be present as an additive during the expression process in order to cleave the shield or decoy proteins from the membrane proteins. An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process. Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or ‘master mix’ which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available. The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the surface by electrokinetic forces by activation of particular electrodes. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. A digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage. Once the CFPS reagents have been enclosed in the droplets, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or ‘power’ sources, or test reagents to which it is desired to expose to the expressed protein. The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil. The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 ^oC. The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the filler fluid can be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of non-movement. Thus the filler fluid can be repeatedly or continually moved for at least a period of 30 minutes or one hour whilst the protein is expressed. The filler fluid can be repeatedly or continually moved for at least a period of two hours whilst the protein is expressed. The filler fluid can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the filler fluid allows mixing within the droplets, and allows oxygen or other reagents to be supplied to the droplets. The act of moving improves the level of protein expression over a system which remains static. Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a- chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense. The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771). A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air/liquid boundary to enable increased diffusion of oxygen from a gaseous environment. Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device. The droplet can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free system having the components for protein expression to form the droplet. The droplets can be split on the device either before, during or after expression. Included herein is a method further comprising splitting the droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one of more of the split droplets are merged with additive droplets for screening. Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin. Optionally fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS production methods. The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPel (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633- 49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275). Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011, 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and/or Pluronic F127. Specifically, droplets containing CFPS components may contain TWEEN 20 at 0.1% v/v, Triton X-100 at 0.1% v/v, and/or Pluronic F127 at 0.05% w/v. For electrowetting on dielectrics (EWoD), the change in contact angle of reagent upon the application of electric potential is an inverse function of surface tension. Thus, for low voltage EWoD operations, reduction in surface tension is achieved by addition of surfactants to reagents, which for CFPS reactions means to the lysate and to the DNA. This results in a dilution of the lysate, and it has been seen, in experiments, that diluting or otherwise adulterating the lysate results in a decrease in expression level of the protein of interest. Thus performing CFPS on DMF where the surfactants are added to the solutions being moved will necessarily result in a dilution and adulteration of the lysate and thus a decrease in the level of protein expression. In addition to being a problem in its own right, this further complicates extrapolation of on-DMF results to in-tube predictions of protein yield. An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to ‘user error,’ as there is more handling of reagents. An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFP11 (or similar) peptide tag, it’s downstream complementation with a GFP1-10 (or similar) detector polypeptide is hindered in the presence of surfactant. Removal of the surfactant from the aqueous phase is therefore advantageous. Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil. This has the advantages of enabling CFPS reactions to proceed on- DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1% w/w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils or blends thereof. Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on-DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split GFP (e.g. GFP₁₁/GFP_1-10) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial. The peptide tag can be attached to the C or N terminus of the protein. The peptide tag may be one component of a green fluorescent protein (GFP). For example the peptide tag may be GFP₁₁ and the further polypeptide GFP_1-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherry₁₁ and the further polypeptide sfCherry_1-10. The protein may be fused to multiple tags. For example the protein may be fused to multiple GFP₁₁ peptide tags and the synthesis occurs in the presence of multiple GFP_1-10 polypeptides. For example the protein may be fused to multiple sfCherry₁₁ peptide tags and the synthesis occurs in the presence of multiple sfCherry_1-10 polypeptides. The protein of interest may be fused to one or more sfCherry₁₁ peptide tags and one or more GFP₁₁ peptide tags and the synthesis occurs in the presence of one or more GFP_1-10 polypeptides and one or more sfCherry_1-10 polypeptides. Devices The manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrokinesis occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semi- conductor film whose electrical properties can be modulated by an optical signal. EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos ^ - cos ^₀= (1/2 ^LG) c.V2 where ^₀ is the contact angle when the electric field across the interfacial layer is zero, ^LG is the liquid-gas tension, c is the specific capacitance (given as ^_r. ^₀/t, where ^_r is dielectric constant of the insulator/dielectric, ^₀ is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant. When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction (ref). The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator/dielectric, (t/ ^)^1/2. Thus, to reduce actuation voltage, it is required to reduce (t/ ^)^1/2 (i.e., increase dielectric constant or decrease insulator/dielectric thickness). To achieve low voltage actuation, thin insulator/dielectric layers must be used. However, the deposition of high quality thin insulator/dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100 V on easily fabricated, thick dielectric films (>3 µm) to effect electrowetting. High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a-Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators/dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator/dielectric devices. Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator/dielectric and a hydrophobic coating or (ii) a hydrophobic insulator/dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator/dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator/dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator/dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. SU-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator/dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or SU-8; thus, the thickness of these materials is typically kept at 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator/dielectric and enable operations with lower applied voltages. Inorganic materials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as “gate dielectrics”, have been used as insulator/dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators/dielectrics), (2) optimizing the fabrication process to make the insulator/dielectric pinhole free to avoid dielectric breakdown. Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator/dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules). One of the adverse effects of this hysteresis is reduced operational lifetime of the EWoD-based device. Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after several operations. The required actuation voltage increases due to this charging phenomenon resulting in eventual catastrophic dielectric breakdown. The most probable explanation is that pinholes at the insulator/dielectric may allow the liquid to come into contact with the electrode causing electrolysis. Electrolysis is further facilitated by pinhole-prone or porous hydrophobic insulators. Most of the studies to understand contact angle hysteresis on EWoD have been conducted on short time scales and with low conductivity solutions. Long duration actuations (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCl) could produce several effects other than electrolysis. The ions in solution can permeate through the hydrophobic coat (under the applied electric field) and interact with the underlying insulator/dielectric. Ion permeation can result in (1) change in dielectric constant due to charge entrapment (which is different from interfacial charging) and (2) change in surface potential of a pH sensitive metal oxide. Both can result in reduction of electrowetting forces to manipulate aqueous droplets, leading to contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces or disables electrowetting on electrodes by inhibiting the modulation of contact angle when an electric field is applied. An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes. The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminium oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 µm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials. The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation/dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 µm thick. The conformal layer may be between 100 nm and 200 nm thick. The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating. The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light. The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination. The electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled. The second substrate may also comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers. The method is particularly suitable for aqueous droplets with a volume of 1 µL or smaller. The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019/0111433, incorporated herein by reference. Described herein are electrokinetic devices, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes; Described herein is an electrokinetic device, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes; The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed. The device can be an active-matrix thin film transistor (AM-TFT) based device. Also disclosed is an active-matrix thin film transistor (AM-TFT) device having a substrate bearing a plurality of electrodes, the device comprising multiple fluidic inlet ports on at least two sides of the device, wherein the inlet ports on each side of the device are evenly spaced and wherein the device is connected to a syringe pump. The device may comprise two substrates, wherein at least one substrate has a plurality of electrodes, and the two substrates define parallel plates that are separated by a spacer to define a volume. The fluidic entry may come via holes in the upper plate or through the spacer. The entry holes may be in the top substrate. The plurality of electrodes may be on a bottom substrate. The top substrate be of glass or polymer and may have a thickness ranging from 0.5 mm to 20 mm. The spacer may comprise an adhesive with beads of a defined size distribution. The spacer may comprise a polymer material of a defined thickness. The spacer may comprise glass, in which case the layers can be fused together. The spacer gap and therefore height of fluid in the device may be between 50 microns and 250 microns. The spacer gap and therefore height of fluid in the device may be between 100 microns and 150 microns. The filler liquid may be moved via an automated manner, or may be moved under gravity. A hydrostatic head of pressure can be used to move the liquid within the device. The wells are at least partially filled with filler fluid before the aqueous reagents are loaded. The filler fluid may be less dense than the aqueous phase such that the aqueous phase sinks in the wells. Alternatively the aqueous phase may sit above the filler fluid, in which case all the filler fluid must be withdrawn from the wells in order to enable entry of the aqueous fluid. The device may be connected to a pump, for example a syringe pump, a peristaltic pump, a disc pump, a diaphragm pump, or a pneumatic pump. The pump enables filling of the device with filler liquid in an automated manner. Once filled, the pump enables partial withdrawal of the filler fluid to create a negative pressure in the device which draws in reagents from the wells. Thus the filling and withdrawal of fluid may be performed in an automated manner to allow largely ‘hands-free’ loading of the aqueous reagents. An automated filler liquid filling and withdrawal method may be integrated into an instrument that provides other functions relating to the digital microfluidic device, including heating, cooling, optical, sensing, mechanical, and magnetic functions. The wells of the device may be at 90 degrees to each other. The inlets may be at 180 degrees to each other. The inlets may be on 4 sides of the device. Each side may have at least 4, 8 or 12 ports. Each side may have 8 ports. The device may have 4 sets of 8 ports. The number of ports may vary on different sides of the device, for example one side may have 8 ports and one side 4 ports. The device may have 8 ports on 3 sides and 16 ports on a fourth side. The ports may be offset to give multiple rows of linear ports on one side, for example a first and second row where the second row is behind by offset from the first row such that the source liquid can flow between the ports of the first row. The rows may be a zig-zag fashion. The pitch between inlet ports may be 9 mm. The pitch between inlet ports may be 4.5 mm. The inlet ports have a pitch of 4.5 mm or a multiple of thereof. This would cover 24 well, 48 well, 96 well, 384 well ports. The pitch of the ports may be the same on each side of the device, or may be different sized. In this context the pitch refers to the distance between the centre of each inlet. The volume of aqueous reagents loaded per inlet port may be between 1 microlitre and 50 microlitres. The volume may be between 1 microlitre and 20 microlitres. The aqueous liquid may be introduced to the wells by a pipette, a multichannel pipette, a syringe, a blister pack, an acoustic dispenser, or a robotic liquid handler. The aqueous liquids may be loaded simultaneously from multiple wells, which may be on the same side or multiple sides of the devices. Each well is a separate liquid, and can be the same or different to the contents of the aqueous volume in other wells. The volume of aqueous liquid loaded in each port can be the same or can be different. The automated filling and/or withdrawing of filler fluid may be controlled by software. The device may be part of a larger instrument system that provides environmental control such as temperature control or light control and may have analytical capabilities such as optical systems for fluorescence or luminescence assay detection. The location of the aqueous layer is controlled by the actuation of electrodes to form reservoirs in defined areas. A plurality of electrodes is actuated to control the location of the aqueous liquid once it has been drawn onto the substrate bearing a plurality of electrodes. Multiple reservoirs may be formed on the device. Examples Comparison of Facade vs Pluronic detergents for reducing hypoxia in aqueous droplets on DMF device via oil sensing.

Level of Hypoxia This example shows the O₂ concentrations present in the device in the presence of a 144- droplet CFPS reaction expressing a green fluorescent protein. The consumption of O₂ causes a hypoxic region which influences the expression/maturation of GFP expressed and appears as a central region of low fluorescence intensity, commonly referred to as a “black hole”. The level of oxygen in the device can be seen using the platinium porphyrin compound Pt(II)OEPK (HSD) in DMPS (50 μg /mL) = HSO, which is added to the basefluid and shows the level of oxygen in the system. The HSO will show a fluorescence intensity in the absence of O₂, and with the timelapse software written for this specific purpose also gives a basis to quantify O₂ consumption and gradient formation. The purpose of this experiment is to map the O2 concentrations in the half-half 144-droplet array of 0.02% Facade TFA1 and 0.05% Pluronic F127 using a cell-free lysate LUPA, which consumes oxygen. This experiment used half LUPA-SBF023 array versus half LUPA-SBF081 array in 144-droplet array with stagnant pattern where the droplets are not circulated during incubation. The HSO oil is used to show a fluorescence intensity in the absence of O2 across the array. Method Prepare 72 µL of the reaction mixture containing LUPA lysate, which already contains T7rnap. The reaction mixture was divided to 2 tubes. The first tube, 0.72 ^L of premix was removed and replaced with 0.72 ^L of 1% TFA1 for a final surfactant concentration of 0.02%, resulting in an overall dilution of 2%. The second tube, 1.8 ^L of premix was removed and replaced with 1.8 ^L of 1% F127 for a final surfactant concentration of 0.05%, resulting in an overall dilution of 5%. 144 droplets of 5x5 pixels are dispensed onto the electrowetting device. The droplets were incubated for 24 h at 29 ^oC using a static mixing pattern. The hypoxia signal began after 6 hours. At 13 hours, the hypoxia fully developed, as shown in Figures 1 & 2. Figure 1 shows the hypoxia after 13 hours, and is greater over the spots having F127. The area of hypoxia intensity was measured and graphed in Figure 2. Between 10 and 13 hours, hypoxia intensity reached a peak and inclined after in both LUPA-surfactants. However, the plot of hypoxia intensity of LUPA-TFA1 was lower, which indicated a smaller black hole. The hypoxia intensity of LUPA-F127 was 2 times greater. Comparison of Facade vs Pluronic detergents for protein expression using LS70 in aqueous droplets on DMF device. Performance of 0.02% Facade TFA-1 (SBF081) and 0.05% Pluronic F127 (SBF023) side- by-side using 144-droplet array running the standard LS70/LEC assay It is shown that SBF081 helps reduce the black hole and increases the expressed protein yield. The array was incubated without droplet movement during expression. Method 96 µL of LS-70 cell-free expression mixture was prepared in order to express fluorescent protein sfGFP_1-11. 24 µL of the reaction mixture was pipetted into a 1.5 mL Eppendorf tube and set aside as a positive, undiluted control, incubated at 29 ^oC.72 ^L was divided into tubes to make 36 ^L each.0.72 ^L of premix was removed from the first tube and replaced with 0.72 ^L of 1% TFA1 for a final surfactant concentration of 0.02%, resulting in an overall dilution of 2%.1.8 ^L of premix was removed from the leftover tube and replaced with 1.8 ^L of 1% F127 for a final surfactant concentration of 0.05%, resulting in an overall dilution of 5%. To prepare 1 mg/mL and 0.5 mg/mL sfGFP standards, sfGFP standard stock at a concentration of 2 mg/mL was diluted with HNG buffer. F127 was added to each for a final concentration of 0.05%. 144 droplets of 5x5 pixels are dispensed onto the electrowetting device. The droplets were incubated for 24 h at 29 ^oC using a static mixing pattern. Figure 3 shows the incubation at 24 hours. The left-hand side of the panel shows the droplets of SBF081. The right-hand side of the panel shows the droplets of SBF023. The fluorescent intensity of SBF081 is uniform across the droplets, which are brighter than the standard 1mg/ml. The black hole is seen here in the right-hand side of the device where the SBF023 located. The liquid contents of the device were removed by draining the cartridge. Figure 4 shows images of the drained device from Figure 3. Residual protein biofouling is greater in the half with F127 and is largely absent in the droplets having Facade. The purpose of this experiment is to compare the O₂ concentrations in the 144-droplet array of 0.02% Facade TFA1 using LUPA/LEC model assay for protein expression. This experiment used a whole 144-droplet array with a standard mixing pattern. The HSO oil is used to show a fluorescence intensity in the absence of O2 across the array. Method showing LUPA expression of sfGFP The expression system LUPA requires a high level of oxygen for expression. The LUPA system continually consumes oxygen, so is highly affected by hypoxia.120 µL of the reaction mixture was prepared containing a e-Coli cell lysate composition having RNA polymerase (LUPA).24 µL of the reaction mixture was pipetted into a 1.5 mL Eppendorf tube and set aside as a positive, undiluted control, incubated at 29 C. 1.92 ^L of premix was removed and replaced with 1.92 ^L of 1% TFA1 for a final surfactant concentration of 0.02%, resulting in an overall dilution of 2%. 144 droplets of 5x5 pixels are dispensed onto the electrowetting device. The droplets were incubated for 24 h at 29 ^oC using a static mixing pattern. The fluorescent signal from the LUPA system was compared to the signal of the LS70 system above. Figure 5 shows hypoxia levels and protein expression comparing SBF081 (0.02% Facade TFA1), SBF023 (0.05% Pluronic F127) and LUPA. Facade TFA1 surfactant has an impact on O₂ consumption or diffusion in a way that reduced the black hole on a device. The area of hypoxia intensity spread 8 droplets horizontally by 8 droplets vertically in SBF023, whereas it only spread 6 droplets horizontally by 6 droplets vertically in SBF081. Standing out, LUPA with TFA1 shows no black hole under GFP and the least hypoxia showed, which is only 3 droplets x 2 droplets. Surfactant comparison for in-tube expression. Objective To investigate the expression of sfGFP protein in direct contact with various surfactants, specifically comparing Pluronic F127 and Facade TFA1. The premix was prepared, split evenly, and subsequently the surfactant introduced at the same dilution factor for each compound. Each premix was transferred to six 1.5 mL eppendorf tubes at different volumes.After 5.5 hours the total fluorescence signal was measured. Method ● Premix was prepared in 1.5 mL Protein Lobind Eppendorf tubes using LUPA E-Coli lysate. ● After surfactant introduction the samples were gently vortexed and spun down to ensure contents were at the bottom of the Eppendorf tube. ● The stocks were then transferred to individual tubes in the volumes indicated below, totalling 12 independent sample tubes. ● The samples were left to incubate in a chamber set to 29 C for 5.5 hours. The incubation chamber was static and no mixing operation was performed. Aqueous phase: 0.05% Pluronic F127 Aqueous phase: 0.05% Facade TFA1 The fluorescence in tubes is shown in Figure 6. Analysis of intensity vs standards of 0.5 and 1.0 mg/mL of sfGFP allows concentrations to be measured. The protein yield of each reaction is shown in the table below and is plotted in Figure 7. LUPA expression is significantly impacted by the amount of available head space in the vessel, likely due to oxygen availability. The Facade surfactant gives a higher yield of protein at all volumes, but is more pronounced at lower reaction volumes. The Facade surfactant appears to allow greater oxygen perfusion in non-mixed systems, including in tubes. The Facade sample reaches its total fluorescence faster than that of Pluronic F127. In larger tube volumes with limited ratio of surface area to atmospheric headspace, the benefits of increased oxygen transfer are reduced. Facade surfactants appear to help protein expression due to an increased oxygen transfer into the aqueous layer. Such findings in both tubes and droplets suggest that tandem facial amphiphiles are useful in a variety of protein expression applications, including in microfluidic droplets. Use of Facade to prevent protein droplet pinning. The aim of this study was to evaluate and compare 2 protein constructs in cell-free expression conditions with and without Facade®-TFA1 detergent. The proteins chosen are known to cause pinning of droplets.5 nM of the nucleic acid template were expressed in a reconstituted cell-free expression system. Images from during the expression are shown in Figure 8, which shows droplets becoming immobilised (pinning) during the expression reaction of both proteins from several timepoints in an expression incubation and the beginning of post-split, illustrating that the droplet pinning without a tandem facial amphiphile started to occur within 1 hour of incubation across the array. Pinning of droplets was observed throughout from expression to incubation, when they were unable to split. However, both constructs did not experience any droplet handling issues in the presence of the tandem facial amphiphile (Facade TFA-1). At the end of complementation, the split without the Facade failed; and droplets remained immobile for the rest of the droplet handling process. Constructs having Facade successfully split into smaller droplets. Columns shown A = Protein 1 in standard expression conditions (No Facade). B = Protein 1 plus Facade. C = Protein 2 in standard expression conditions. D = Protein 2 plus Facade. Columns A and C have failed to split into smaller droplets. Columns B and D behave as expected. The images show the addition of Facade prevents the protein droplets becoming immobilised on the device surface. Figure 9 shows fluorescent of the device after complete draining of the liquids. The areas showing fluorescent signal are deposited protein (corresponding to columns A and C in the images of Figure 8). The presence of Facade largely eliminates the protein fouling (Columns B and D), which is only visible in columns where the Facade is absent.

Claims

Claims 1. A method comprising the use of a tandem facial amphiphile for cell-free protein synthesis. 2. The method according to claim 1, wherein the cell-free protein synthesis is performed in aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. 3. The method according to claim 2, wherein the droplets are in an aqueous immiscible filler fluid. 4. The method according to any one of claims 1 to 3, wherein the tandem facial amphiphile is a compound of formula (I): wherein L is —(CH2)n— where n is 1-10; (C5-C8)cycloalkyl; a phenyl diradical optionally substituted by 1, 2, 3, or 4 (C1-C4)alkyl groups; —C(R^x)2—; or —CH2—C(R^x)2—CH2—; where each R^x is independently H, OH, or —CH₂O-Sac; each X is independently O, S, NH, CH₂, triazole, or a direct bond; each m is 0 or 1; each R¹ is independently H or (C1-C20)alkyl; and each Amph is independently a moiety of Formula A:

wherein Y is CH2 or a direct bond; each R², R³, and R⁴ is independently H, OH, or O-Sac; and each Sac is independently an oxygen-linked monosaccharide, disaccharide, or trisaccharide where the compound of Formula I has at least 4 Sac groups. 5. The method according to claim 4, wherein each sac is a disaccharide. 6. The method according to claim 4 or claim 5, wherein X-L-X is —(CH2)n— where n is 3- 10. 7. The method according to any one of claims 4 to 6, wherein R1 is CH3. 8. The method according to any one of claims 4 to 7, wherein each m is 1. 9. The method according to any one of claims 4 to 8, wherein each R₂ and each R₃ are disaccharides. 10. The method according to any one preceding claim, wherein the tandem facial amphiphile is selected from:

11. A method according to any one of claims 2 to 10 for the synthesis of a protein in aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising mixing droplets having a reaction system with at least one template nucleic acid encoding a protein of interest and droplets having a cell-free expression system including enzymes for protein synthesis, wherein either or both of the droplets contain one or more tandem facial amphiphiles. 12. The method according to any one of claims 2 to 10, wherein the aqueous droplets contain a fluorescent protein whose fluorescence intensity is oxygen dependent.

13. The method according to any one of claims 1 to 12, wherein the cell-free system is a lysate is derived from mammalian cells prokaryotic cells, yeast cells, plant cells or protozoa. 14. The method according to claim 13, wherein the cell lysate is derived from Escherichia coli. 15. The method according to claim 14, wherein the lysate is supplemented with one or more reagents selected from synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, a polyethylene glycol, an allolactose, an aldohexose or a thiogalactopyranoside, buffers, surfactants, metal ions, chaperones, co-factors or additional protein components. 16. The method according to claim 15, wherein the additional protein components are selected from chaperones, glycosylating enzymes, proteases, redox active enzymes, N-acetyl transferases, phosphorylases and kinases. 1 The method according to any one of claims 1 to 16, wherein the cell-free protein synthesis produces a membrane protein. 18. The method according to any one of claims 1 to 17, wherein the cell-free protein synthesis produces a protein having a solubility tag. 19. The method according to any one of claims 1 to 18, wherein the cell-free protein synthesis produces a protein having a detection tag which is a component of a fluorescent protein. 20. The method according to any one of claims 2 to 19, wherein the digital microfluidic device is an active-matrix thin film transistor (AM-TFT) based device. 21. The method according to any of claims 2 to 20, wherein the filler fluid is dodecamethylpentasiloxane, decane or dodecane. 22. The method according to any of claims 2 to 21, wherein the filler fluid contains a surfactant. 23. The method according to claim 22, wherein the surfactant is a sorbitan ester.

24. A kit comprising a tandem facial amphiphile in a cell-free protein expression reagent, an aqueous immiscible filler fluid and an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. 25. A kit according to claim 24, wherein the tandem facial amphiphile is selected from

or a combination thereof. 26. The method or kit according to any one preceding claim wherein the concentration of the tandem facial amphiphile is at an amount of 0.01 to 0.1 % by volume.