EP4633809A1

EP4633809A1 - Gas exchange during electrowetting operations

Info

Publication number: EP4633809A1
Application number: EP23828779.1A
Authority: EP
Inventors: Maheshwar ADIRAJ IYER; Sumit KALSI; Johnathon LILLEY; Gordon Ross MCINROY; Luke M. SLOMINSKI; Andreas Michael WAEBER; Andrea Zanzotto
Original assignee: Nuclera Ltd
Current assignee: Nuclera Ltd
Priority date: 2022-12-16
Filing date: 2023-12-18
Publication date: 2025-10-22
Also published as: WO2024127038A1; GB202219012D0; CN120359086A

Abstract

Provided herein are methods for replenishing dissolved gases to aqueous droplets on digital microfluidic devices. Provided herein are methods of cell-free protein synthesis, optimised cell- free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by circulating the filler fluid in order to introduce additional oxygen.

Description

GAS EXCHANGE DURING ELECTROWETTING OPERATIONS

FIELD OF THE INVENTION

Provided herein are methods for replenishing dissolved gases to aqueous droplets on digital microfluidic devices. Provided herein are methods of reducing hypoxia 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

The handling of small volumes of reagents is facilitated using droplet based microfluidics, for example digital microfluidic based systems involving electrowetting. However the use of small reagents volumes can lead to the rapid consumption of dissolved gases such as for example oxygen or carbon dioxide which may be required for particular applications in the droplets.

The growth of cells for example may rely on a supply of particular gases such as for example carbon dioxide.

Cell-free protein synthesis (CFPS) has 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 (Langmt//r2011 , 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 have appreciated that the isolated aqueous droplets are rapidly depleted of dissolved gases, and that dissolved gases need to be replenished. The inventors herein have improved the uniformity of CFPS systems and cell growth systems on electrowetting devices using methods to supplement gases from the filler fluid to the droplets.

SUMMARY

In aqueous droplets on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes the supply of dissolved gases such as oxygen or carbon dioxide 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. The growth of cells requires carbon dioxide or other dissolved gases. Disclosed herein are methods for replenishing dissolved gases by circulating the base fluid within an electrowetting-on-dielectric (EWoD) device. Disclosed herein are methods for retaining or increasing hypoxia by replenishing carbon dioxide to aqueous droplets.

Disclosed herein are methods for decreasing hypoxia in aqueous droplets in an aqueous immiscible filler fluid on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes. Disclosed herein are methods for introducing additional oxygen into aqueous droplets on a digital microfluidic device having a plurality of electrodes. The additional oxygen can be introduced by moving a filler fluid in relation to the aqueous droplets and/ or by moving droplets in relation to the filler fluid. The droplets can be held whilst the filler fluid is replenished.

Disclosed is a method for replenishing gas to aqueous droplets in an aqueous immiscible filler fluid on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising holding the droplets and moving the filler fluid in order to replenish gas to the aqueous droplets, wherein a portion of the filler fluid is withdrawn from the device, thereby introducing filler fluid having a higher level of dissolved gas.

Disclosed is a method for the synthesis of a protein in aqueous droplets in an aqueous immiscible filler fluid 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 lysate including enzymes for protein synthesis and moving the filler fluid in order to replenish oxygen to the aqueous droplets.

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 dissolved gas than the fluid which has been withdrawn. The fluid may be withdrawn using an automated flow or under gravity. The fluid may be recirculated in order to minimise the waste of filler fluid. The filler fluid may contain the dissolved gas such as oxygen naturally. The filler fluid may be re-gassed by exposing to an atmosphere rich in the desired gas, for example by bubbling the gas into the filler fluid. Where the oxygen is used, atmospheric air would also be applicable to replenish the oxygen in the filler fluid. Thus the filler fluid may be oxygenated or re-oxygenated by stirring or bubbling air through the oil for example.

Alternatively or additionally to flowing the oil, the aqueous droplets may be moved within the device, as disclosed in application W02021/161048. 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 W02021/161048 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 crosscontamination 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 oxygen is introduced during the expression to increase the yield of protein.

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 or protozoa. The cell lysates may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa, BHK21 , NSO, 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 GFPn and the further polypeptide GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherrymo. The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

For example, the GFP1.10 polypeptide amino acid sequence could be derived from sfGFP:

MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSTQSVLSKDPNEK

Alternatively, the GFP1.10 polypeptide amino acid sequence could be further mutated from the sequence above to become brighter more quickly upon complementation:

MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVT TLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAWKFEGDTLVNRI ELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSTQTVLSKDPNEK

The GFPn peptide amino acid sequence should be of sufficient length to bind to GFPi- and produce a fluorescence signal. The complementary GFPn peptide amino acid sequence could be the following: KRDHMVLLEFVTAAGITGT

KRDHMVLHEFVTAAGITGT

KRDHMVLHESVNAAGIT

RDHMVLHEYVNAAGIT

GDAVQIQEHAVAKYFTV

GDTVQLQEHAVAKYFTV

GETIQLQEHAVAKYFTE

GFPn or GFP1.10 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 sfCherryi- polypeptide amino acid sequence could be:

MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDI LSPQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGGVVTVTQDSSLQDGEFIYK VKLLGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVDIKLDITSHNED

The complementary sfCherryn peptide amino acid sequence could be:

YTIVEQYERAEGRHSTGG sfCherryn or sfCherryi- 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:

MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKQVIGKN FFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKVKVHMKKALS

The complementary CFASTn peptide amino acid sequence could be:

GDSYWVFVKRV

Or the complementary CFAST10 peptide amino acid sequence could be:

GDSYWVFVKR NFAST, CFASTn, 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 sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi- polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1.10 polypeptides and one or more sfCherryi- 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 Polp, Poip, PolA, and PolQ of any species or the homologous amino acid sequence of X family polymerases of any species.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an image of aqueous fluid being loaded into a digital microfluidic device seated in an instrument. The aqueous fluid loading is facilitated by the presence of an automated syringe pump that can add or remove filler fluid at programmably defined flow rates. The syringe pump primes the device with filler fluid and also assists with the loading of aqueous reagents and filler fluid from the loading wells by withdrawing the filler fluid from opposing ports.

Figure 2 shows a diagrammatic figure of the loading process.

Figure 3 shows oxygen depletion (hypoxia) seen during protein expression, image of GFP expression in droplets and hypoxia layers in a 144-droplet array. Figure 4 shows the linescan and overlap between reaction zones and hypoxia map based on Figure 3.

Figure 5 shows the slow recovery by simple diffusion. After 18 hours, little recovery of the oxygen levels are seen.

Figure 6 shows schematic representation of actuation of static and circulating dummy droplets.

Figure 7 shows the development of hypoxia is mitigated by circulating dummy droplets.

Figure 8 shows the hypoxia after 18 hours and a graphical depiction.

Figure 9 shows four cross sections of signal. Row 3 is the centre of the static zone. Row 10 is the centre of the circulating zone. Row 10 shows the most consistent intensity with minimal reduction due to hypoxia.

Figure 10 shows the effect of circulating droplets of varying size.

Figure 11 shows a linescan of the images in Figure 10.

Figure 12 shows an oil refresh experiment performed at 29 °C. A complete oil refresh was carried out after 3 hours of total assay incubation time (including loading, dispensing, and on- device incubation). The oil refresh was carried out using a syringe-pump, using diagonal ports to exchange 2 mL of oil in 4 minutes at a flowrate of 500 pL/min while all aqueous droplets are being held in place by continuous actuation. After oil refresh was complete, the assay was allowed to go to completion without further intervention. The timing of the oil exchange placed it approximately midway through the exponential phase of protein expression. Figure 12 shows the comparison with and without the oil exchange.

Figure 13 shows analysis of the data from Figure 12. The oil exchange pre-empted much of the hypoxia and led to a higher overall yield and significantly lower variability between droplets.

Figure 14 shows that refreshing oil lowers the levels of hypoxia. Oil refresh was carried out after 5 hours of assay time at 20 °C, before hypoxia developed on the device (hypoxia typically observed after 5-6 hours of incubation time). The assay was allowed to incubate at room temperature (20 °C) until total assay time was 24 hours. Hypoxia began to appear at ~ 13 hours of incubation time in the oil refreshed images and 7 hours in the baseline system. Figure 15 shows images continued from Figure 14 at 24 hours incubation. The hypoxia sensor showed a hole in the centre of the device, but the fluorescence spots from protein expression are uniform with no evidence of hypoxia, thus the oil refresh after 5 hours improves the fluorescence signal from expressed protein.

Figure 16 shows an incubation script showing the two halves of the device. The standard mixing pattern is used on the right, while droplets move along a continuous serpentine loop pattern on the left.

Figure 17 shows an image of the device taken after 22 hours of assay time. A single pinned droplet can be seen in the center of the device. A black hole has formed in the right-hand side of the device where the typical mixing pattern was applied, while the left-hand side of the device, on which the droplets travelled in the serpentine loop pattern, shows no such pattern. It can be seen that the droplets on the serpentine side look relatively uniform in fluorescence intensity compared to the right-hand side.

Figure 18 shows the heatmap of Figure 17. The average yield on the device side that was running the typical mixing pattern is 1.21 mg/mL, with a CV of 47.8% (very large as expected, due to development of black hole). On the side of the device on which the serpentine loop ran, the average droplet yield is 1.45 mg/mL, with a CV of 6.8%. The movement of the droplets around the array seems to have evened out the effects of location, in particular oxygen availability. In addition to reducing the fluorescence variability between the droplets, the average yield per droplet has also increased relative to the side on which oxygen limitations existed toward the center of the device.

Figure 19a shows a diagrammatic figure of the loading process (shown for a single port). The device is made from a bottom plate having an array of electrodes (TFT), a top plate (optionally glass although may be plastic) defining a cell gap therebetween. The top glass contains holes for loading reagents. The top glass holes are located underneath a plastic housing containing inlet loading ports to load regents into the cell gap. (A) A device is filled with filler fluid to fill the cell gap and at least partially fill all the inlet ports. (B) Reagents are placed into the ports using an external source, for example a multi-channel pipette (one channel shown). (C) The reagent sinks to the bottom end of the port, close to the entry hole in the top glass. (D) Filler fluid is removed from the cell gap and the reagent is introduced into the device for downstream operations. Figure 19b shows a diagrammatic figure of a device, with one port connected to the filler fluid withdrawing unit and ports available for loading aqueous reagent. The loading process and subsequent filler fluid withdrawal can be driven by removing filler fluid from one port or multiple ports. The multiple ports can be placed on the same side or on opposite sides on the device.

The oil can be extracted from two corners of the device which can be on the same side or diagonal corners. The fluid used to replenish the gas can be drawn in via the filled loading ports by withdrawing fluid from opposing ports. The removal can be performed using a pump in order to produce a negative pressure (i.e. the pump can push the oil into the microfluidic gap and fill the loading ports, then pull using a negative pressure to introduce fresh filler fluid from the loading ports into the microfluidic gap).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are methods of replenishing gases to aqueous droplets. The gases may be replenished by circulating the filler fluid, either internally or by replenishment from external sources.

Disclosed is a method for growing cells where hypoxia conditions are maintained by replenishment with oil containing dissolved carbon dioxide.

Disclosed is a method for decreasing hypoxia in aqueous droplets in an aqueous immiscible filler fluid on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising 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.

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 (DM PS). 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 (R SC 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, cofactors, 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., EB PKA)

■ Protease 1 (e.g., NEB TEV)

■ Protease 2 (e.g., Merck HRV 3C)

■ 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.

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 °C.

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 ( . 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 FluoroPei (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 GFPn (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/GFP1.10) 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 GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- .

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 sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi- polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1.10 polypeptides and one or more sfCherryi.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 semiconductor film whose electrical properties can be modulated by an optical signal.

EWoD phenomena occur when droplets are actuated between two 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: cosQ - cos0o= (1/2yLG) c.V² where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as £_r. £o/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. 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/£_r)^1/2. Thus, to reduce actuation voltage, it is required to reduce (t/e_r)^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 pm) 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. Sll-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 Sll-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 NaCI) 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 pm 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 pm 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 pL 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 and filler fluid 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/loading ports 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/loading ports 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.

The removal can be performed using a pump in order to produce a negative pressure (i.e. the pump can push the oil into the microfluidic gap and fill the loading ports, then pull using a negative pressure to introduce fresh filler fluid from the loading ports into the microfluidic gap). IN such example the filler fluid goes throOugh the device to the loading ports and re-enters the device from the loading ports to replenish the filler fluid in the microfluidic gap in which the dissolved gases have been consumed.

Examples

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 platinum porphyrin compound Pt(ll)OEPK (HSD) in DMPS (50 pg /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.

Methods

The experiment is designed as a layer over a standard CFPS reaction. The DMPS oil phase is supplemented with HSO, which is a 1 mg/mL stock solution of HSD in C12 diluted 20x to 0.05 mg/mL in DMPS. CFPS premix with 0.05% F127 is loaded into reservoirs and dispensed into the HSO oil. The experiment then continues as a conventional CFPS reaction, with the mixing occurring in a figure-8 pattern overnight.

Trial-2 dispensed an expression panel of 144 droplets to push the formation of the hypoxia black hole. The hypoxia dye is imaged using the NIR filter and UV LED. A 5-second exposure is used to capture any weak fluorescent signal from the hypoxia dye, for 24 h. At the end of the experiment, the NIR filter is replaced with the 550 nm filter to image the final protein expression screen. A composite pseudochrome image can then be built from the NIR channel and the green channel to provide a complete perspective of the O2 content vs the expression of protein in the device. The aim of this overlay is to validate the hypothesis that the dip in protein expression is caused due to a reduction in O2, which should be indicated by the fluorescence of the HSO overlapping spatially with the dip in fluorescence of the protein. Lower oxygen levels in the centre of the device lower the expression of the protein.

Results

The HSO emits a fluorescence signal in the absence of O2, and thus is an indicator of hypoxia. The earliest a faint signal can be detected is around the 5 - 5.5h mark into the CFPS reaction. The dark structures seen in the image are the CFPS droplets. The HSD is dissolved in the oil, with excitation at 395 nm and detection through an NIR filter at 750 nm; and hence cannot be visualized simultaneously with the protein expression. The droplets are actuated in a mixing pattern which leaves them misaligned with the grid layout when they pin on the device.

The hypoxia signal originates from roughly the center of the device. Over time, it spreads outwards from the center of the device, which is also expected as hypoxia would occur in the regions farthest from the porting holes that are exposed to the ambient atmosphere and hence can act as a secondary O2 source. The CFPS reactions occurring in the center of the device act as an O2 sink, and the process could be diffusion-limited between the O2 supply from the porting hole and the consumption at the CFPS droplets.

A fluorescence image was captured with the filters changed for GFP, and was overlaid with the HSO mapping to give a pseudochrome image with a GFP layer and a hypoxia layer (Figure 3). The hypoxia band overlaps well with the black hole, and is expected to be an important contributor to the fluorescence non-uniformity. The green and the red spectral overlap shows quite distinctly that the hypoxic region is present in the center of the device and grows outwards with increasing time intervals. Figure 4: Linescan and overlap between reaction zones and hypoxia map. The fluorescence signal from the hypoxia overlaps with the location of the low-expressing CFPS reaction zones. The black hole formation in the 144-droplet panel is prominent enough to be detected visually on a fluorescence image captured of the panel.

Figure 5: Recovery from hypoxia after conclusion of GFP-CFPS. The outer trend line is a linear profile taken soon after the CFPS reaction is concluded and all the data from the reaction is acquired. The incubation phase is continued under the premise that O2 would diffuse through the porting holes and cause the oxygen to bounce back, thus quenching the dye. Over an 18- hour observation window, there is some reduction in hypoxia, seen by the shrinking trendline for the HSO linescan. However even after 18 hours, the centre of the device remains hypoxic and lacking in oxygen.

Use of circulating droplets to reduce hypoxia

This experiment incorporates the use of dummy droplets to achieve circulation of the oil on the device by moving through and displacing the oil between the reaction zones to bring in O2- rich oil from the outer regions of the panel towards the middle.

A 12x12 array of GFP-CFPS droplets was dispensed, followed by a 7x12 array of dummy droplets that occupied the voids between the CFPS reaction zones. The dummy droplets are actuated to circulate around a row of CFPS reaction zones. This pattern is used on one side of the panel, with the other half of the panel being held with static incubation (Figure 6).

Development of hypoxia was monitored under a Basler-NIR camera with images captured at 15 minute intervals for 24 hours. At the end of the 24-hour period, the panel was also imaged under a Sony DSLR camera to check the expression. The images can then be overlaid in pseudochrome channels to visualize the hypoxia and expression maps at the same time. Temporal trends can be picked up with these timelapse images and analysis with Imaged (Figure 7).

Hypoxia was seen to develop around the 5 hour mark, and developed in the side with static incubation earlier than the side with the circulatory droplets. Movement of the oil also delayed the development of hypoxia due to the constant movement of the droplets and the displacement of the oil on the panel. The dummy droplets cause the apparent movement of oil between the 02-rich edges and the 02-depleted interior of the droplet array. The stripes in the hypoxia profile indicate the apparent movement of oil between these regions, with the darker regions being richer in O2 and the brighter regions having a lower O2 content, leading to the fluorescence of the HSO. The alternating bright and dark stripes also suggest the circulatory movement of the droplets brings in the oil from the edges (detected as the dark stripe, moving inwards) and displaces the O2-depleted oil towards the outer edges (detected as the bright strips, moving outwards). Figure 8 shows linescans through the sections of static and circulating droplets and showed a higher signal for the reaction zones in the static incubation section vs the hypoxia in the dummy droplets section.

The protein expression showed the formation of a black hole effect in the center of the device that overlapped with the O2 depleted region in the static section. The section with the dummy droplets also showed the formation of a black hole, but the fluorescence intensity was higher than the corresponding positions in the static incubation section. The section with missing droplets however, showed a fluorescence intensity that was closer to the black hole than the section with uninterrupted circulation (Figure 9). The fluorescence intensities of the droplets in Row-7 (circulation) were more than the intensities of the droplets in Row-6 (static). Rows 3 and 7 can be compared, with each row equidistant from static-circulation boundary on the panel. The fluorescence intensity of the droplets in Row-7 (circulation) was significantly higher than the intensity of the droplets in Row-3 (static), indicating that the movement of oil increased the O2 availability in the vicinity of the reaction zones, leading to higher protein yield.

Conclusion

The looping movement pattern of the dummy droplets replenishes O2 content of small volumes of oil and displaces this 02-rich oil into the 02-depleted center of the device. Higher protein yield indicates the successful increase in O2 availability due to the oil displacement as a result of the movement of the dummy droplets.

Testing sizing of circulating droplets

This experiment aims to test the influence of droplet size on the bow-wave effect as seen previously. The term bow-wave effect is used as a hypothetical approximation of the displacement of oil by the movement of aqueous droplets inside the DMF device. The premise of this experiment is that the displacement of the oil (and hence hypoxia mitigation) will be magnified if the droplets being actuated are larger in size.

Methods

Droplets of GFP-CFPS premix and circulatory dummy droplets were dispensed from reservoirs located directly opposite each other. The locations of the droplets were staggered at dispense, and movement on the panel was restricted to being in one dimension to avoid collisions between the dummy droplets and the CFPS premix. 36 droplets of size 100 were dispensed and split twice in succession to form a 12x12 array of CFPS premix. The voids between those droplets were occupied by the larger dummy droplets, which were dispensed as sz100 droplets but split only once to maintain them at 7x7 dimensions. Droplets any larger in any direction would collide into the CFPS array, so 7px is used as the maximum length of a droplet dimension in conjunction with a 12x12 CFPS array with a 25px spacing. Larger droplets are only recommended with arrays with larger spacing.

Pathing was scripted such that the GFP-CFPS premix and the dummy droplets are always parallel to each other in opposite directions and never on intersecting paths. The preferential path for this movement was horizontally, between E2 and E1. All vertical movements were finished after dispense but before the droplets entered the active area of the droplet array, purely to avoid any potential collisions during droplet dispense and positioning.

Timelapse imaging was done using timelapse software for 24 hours, and final protein expression was measured via fluorescence using the DSLR at 550 nm emission.

Results

The movement of HSO in the device can be seen on the circulating side as bright and dark stripes. The bright stripes are the O2-depleted HSO being displaced towards the outside, and the dark stripes are the 02-rich oil being displaced towards the hypoxic center. The stripes are notably weaker in the sz49 trial than the stripes in the sz25 trial. The larger sized droplets would also displace a greater volume of oil during movement, which would help reduce the O2 gradient, which is visualized as the intensity of the oil in the stripes of oil being displaced (Figure 10). The hypoxia profile of the panel with the circulating sz49 dummy droplets was notably lower than that of the sz25 dummy droplets, due to the probable higher displacement of the oil (Figure 11). The low hypoxia signal from the circulating dummy droplets section for sz49 droplets also suggests that the oil displacement occurring due to the constant movement replenishes the O2 in the central regions of the panel such that significant O2 depletion is prevented. Comparing fluorescence intensities between the static side vs the circulating dummy droplet side of the panel, there is an observable formation of the black hole on the static incubation side, while the other half of the panel with the circulating dummy droplets showed no appreciable drop in fluorescence between the droplets.

Conclusion

The difference in fluorescence intensities between the side with static incubation and the side with dummy droplets agrees with earlier experiments involving movement of the reaction zones as well as dummy droplets improving the oxygen availability in the device. The larger dummy droplets also showed a lower hypoxia signal, suggesting that the effectiveness of hypoxia mitigation would be directly related to the displacement of oil between the normoxic and hypoxic zones.

Testing Oil Replenishment

The objective of this experiment was to further investigate the feasibility of using a dual-syringe pump method of simultaneous infusion/withdrawal of carrier oil to accomplish an oil refresh on DMF as a method of increasing available oxygen. To generate the array of droplets, LS70 lysate containing 0.05% F127 was loaded into the DMF cartridge using a syringe pump. No other reagents were loaded as an array of 108 size 7 droplets (since no DNA was added). Once an array of droplets had been generated on the device, two identical Chemyx syringe pumps, with accompanying tubing, were set up to input and output oil to/from the cartridge at the same flow rate. With the two syringe pumps set to infuse and withdraw oil simultaneously, the flowrate of oil, as well as the droplet mixing pattern, were varied to determine the maximum flowrate that would be tolerated before the droplets were perturbed, as well as to determine whether certain actuation modes (i.e. mixing vs stationary) had an effect on droplet stability.

Results

The oil flowrate was set to 0.1 mL/min and the effect on droplet movement was monitored. At this flowrate, no effect on droplet movement was observed. The flowrate was then increased to 0.3 mL/min, 0.5 mL/min, 0.7 mL/min, and 1 mL/min. No effect on droplet movement was observed at any flowrate up to and including 1 mL/min (the highest flowrate used). Also of note, there was neither spillage of oil out of the ports, nor air bubble formation observed, meaning that the flow in and out of the device was balanced (i.e. the volume within the device was at steady state). This flowrate is also considerably higher than what would most likely be used during an assay for either one-time or continuous oil exchange, so this effectively means that the flowrate will not be a constraint in further development of this method.

Testing Oil Replenishment for Protein Expression

The goal of this experiment was to test a single time point oil refresh during an on-device assay run to determine its effect on GFP expression yield and variability. It is hypothesized that replacing the oxygen-depleted oil on the device with fresh oil at a point in the assay at which hypoxia is expected to first develop but before it begins to inhibit protein expression, i.e. at some point during the exponential protein expression phase, will lead to higher overall protein yield with a minimized (or eliminated) black hole. This would be reflected in a higher average protein yield accompanied by a lower %CV across the device. 144 Droplets of GFP-CFPS premix were dispensed from reservoirs. An oil refresh was carried out after approximately 3 hours of total assay time from the time the premix was prepared intube (this included the time it took to load, dispense, and form the array, approximately 1 hour and 20 minutes). This time was selected based on previous experiments, and coincides with the time frame during which protein expression is seen to be proceeding rapidly, but before a discernible black hole has developed.

The oil refresh step was carried out with an oil flowrate of 0.5 mL/min, using diagonal ports for simultaneous infusion and withdrawal. After this single time point refresh, the assay was left to incubate without further interference for a total assay time of 24 hours. The oil refresh step did not perturb any of the droplets or remaining reservoirs on the device.

Figure 12 shows an image of the device at the end of the experiment. Fluorescence intensity appears uniform and a significant black hole is not evident, unlike the control where the oil is not refreshed. The figure shows a comparison of the final images and heatmaps of the oil refresh experiment compared to baseline. In the figure, the same conditional formatting rule was applied to both heatmaps of protein yield. A faint black hole is visible on the oil refresh heatmap of yield, in the same shape as the one that appears on the yield heatmap of the baseline run. This suggests that some minor hypoxia is still present, and the assay would likely benefit from additional oxygenation. This could be in the form of either an additional oil refresh step (or changed timing of the single exchange) or continuous oil refresh at a flowrate sufficient to fully eliminate hypoxia. The average on-device yield was calculated to be 1.87 mg/mL with a CV of 4.0%. In the absence of oil exchange, the average on-device yield was 1 .43 mg/mL with a CV of 30.1%.

Figure 13 shows quartile boxplots of assay yield from the baseline ACA run versus the ACA run on which the oil refresh was executed. The higher average yield and tighter spread of the run which contained the oil refresh step is evident.

HSO test

The goal of this experiment was to test a single time point oil refresh in the presence of hypoxia-sensing oil (HSO) at room temperature to determine the effect of the oil refresh on the timing and extent of hypoxia development compared to a baseline experiment in which an oil refresh was not performed.

144 Droplets of GFP-CFPS premix were dispensed from reservoirs. An oil refresh was carried out after 5 hours of total assay time from the time the premix was prepared in-tube. This time was selected to fall before the appearance of on-device hypoxia, based on previous experiments. The oil refresh step was carried out with an oil flowrate of 0.5 mL/min, using diagonal ports for simultaneous infusion and withdrawal while all droplets were actuated in place. A total volume of 2 mL of oil was replaced. After the refresh, the assay was left to incubate without further interference for a total assay time of 24 hours.

Figure 14 shows time lapse images from the cartridge at 5, 10, 13 and 15 hours. During the baseline run, hypoxia began to develop after 7 hours and was almost fully-developed after 11 hours of assay time. By comparison, during the oil refresh run hypoxia began to develop only after 13 hours.

Figure 15 shows an image of the device after 24 hours. Several dud droplets are visible. At the end of the assay, the on-device fluorescence intensity looked uniform, and no significant black hole was seen to have formed. Average on-device yield was calculated to be 1.66 mg/mL with a CV of 6.6%. A similar baseline run of the standard assay on ACA showed a yield of 1.37 mg/mL, with a %CV of 15.5%, demonstrating improved yield with lower variability for the assay with the oil refresh step.

An analysis of the time lapse hypoxia images showed that hypoxia developed later on the device with an oil refresh vs. baseline (13 hours vs. 5 hours of assay time, respectively). The development of hypoxia, albeit at a late stage, suggests that the assay may benefit from further additional oxygenation to potentially further increase protein yield.

Loop mixing

The goal of this experiment was to compare two different mixing strategies side-by-side using the 144-droplet array running the standard assay. A mixing pattern that uses a command for near-continuous mixing was used on one half of the device, while the other half of the device ran a full-side serpentine loop pattern in which droplets moved continuously along a circuitous path that encompassed that entire half of the array, such that every droplet spent near-equal time in every droplet location. The objective was to eliminate spatially-dependent yield patterns, such as the black hole, that form when droplets remain in their initial locations throughout an experiment and become subject to location-dependent variables such as oxygen availability and local variations in temperature.

144 Droplets of GFP-CFPS premix were dispensed from reservoirs. Incubation was then initiated. For the incubation phase, the device was divided into halves. Incubation on one half of the device consisted of running the typical mixing script, which consists of almost- continuous mixing. On the other half of the device a serpentine travel pattern for the droplets was initiated. This pattern moves ali droplets along a path that repeatedly criss-crosses the device. The purpose of this travel pattern is to average out all spatial effects of yield, including oxygen availability, so as to equal out the yield achieved by all droplets. In the current form of the script, each loop of the script moves each droplet by one location. As a result, it takes 72 loops of the script to move the droplets through a full path around the device. Given the number of steps in the script and the current frame rate, the entire pattern takes - 100 minutes for each droplet to complete. The division of the device into halves and the serpentine pattern formed by the script are shown in the figure below (Figure 16).

Figure 17 shows an image of the device taken after 22 hours of assay time. A single pinned droplet can be seen in the center of the device. A black hole has formed in the right-hand side of the device where the typical mixing pattern was applied, while the left-hand side of the device, on which the droplets travelled in the serpentine pattern, shows no such pattern. It can be seen that the droplets on the serpentine side look relatively uniform in fluorescence intensity compared to the right-hand side.

Figure 18 shows the average yield on the device side that was running the typical mixing pattern is 1.21 mg/mL, with a GV of 47.8% (very large as expected, due to development of black hole). On the side of the device on which the serpentine ran, the average droplet yield is 1.45 mg/mL, with a CV of 6.8%. The movement of the droplets around the array has evened out the effects of location, in particular oxygen availability. In addition to reducing the fluorescence variability between the droplets, the average yield per droplet has also increased relative to the side on which oxygen limitations existed toward the center of the device.

Also of note, the fluorescence intensities of the serpentine droplets are, on average, higher than those that were mixed using the typical mixing pattern. However, the highest fluorescence levels observed along the edges (outside) of the typical side of the device are higher than the levels observed among serpentine droplets. This suggests that if more oxygen could be provided to the serpentine, then this level of yield could be (uniformly) achieved by all of the droplets. That is, although the serpentine movement pattern has helped to equalize and optimize consumption of the available oxygen by the droplets, there is still an inherent oxygen limitation present that is preventing the droplets from reaching the highest levels of yield seen on the black hole side of the device.

Claims

1 . A method for replenishing gas to aqueous droplets in an aqueous immiscible filler fluid on an electrowetting-on-dielectric (EWoD) device bearing a plurality of electrodes, the method comprising holding the droplets and moving the filler fluid in order to replenish gas to the aqueous droplets, wherein a portion of the filler fluid is withdrawn from the device, thereby introducing filler fluid having a higher level of dissolved gas.

2. The method according to claim 1 wherein the gas is carbon dioxide or oxygen.

3. The method according to claim 1 for the growth of cells in droplets on the device wherein the droplets are replenished with carbon dioxide.

4. The method according to claim 1 for the synthesis of a protein in aqueous droplets in an aqueous immiscible filler fluid 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 lysate including enzymes for protein synthesis and moving the filler fluid in order to replenish oxygen to the aqueous droplets by introducing filler fluid having a higher level of dissolved oxygen.

5. The method according to claim 1 or claim 4, wherein the aqueous droplets contain a fluorescent protein whose fluorescence intensity is oxygen dependent.

6. The method according to claim 5, wherein the protein droplets are stationary whilst the filler fluid moves.

7. The method according to any one of claims 4 to 6, wherein the cell lysate is derived from mammalian cells prokaryotic cells, yeast cells, plant cells or protozoa.

8. The method according to claim 7, wherein the mammalian cells are HEK293, HeLa, BHK21, NSO, Sp2/0, or CHO.

9. The method according to claim 7, wherein the cell lysate is derived from Escherichia coli.

10. The method according to claim 7, wherein the cell lysate is derived from Saccharomyces cerevisiae or Pichia pastoris.

11. The method according to claim 7, wherein the cell lysate is derived from tobacco or wheat.

12. The method according to claim 7, wherein the cell lysate is derived from Leishmania tarentolae.

13. The method according to any one of claims 4 to 12, 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.

14. The method according to claim 13, wherein the additional protein components are selected from chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

15. The method according to any one of claims 4 to 14, wherein the reagent composition is formed on the EWoD device by merging a first droplet containing a cell lysate and a second droplet containing a template nucleic acid and optionally a third droplet having an additional component.

16. The method according to any one preceding claim, wherein the electrowetting-on- dielectric (EWoD) device is an active-matrix thin film transistor (AM-TFT) based device.

17. The method according to any one preceding claim, wherein the filler liquid is drawn from the device under gravity or by a syringe pump, a peristaltic pump, a disc pump, a diaphragm pump, or a pneumatic pump.

18. The method according to any one preceding claim, wherein the filler fluid is used to fill loading ports on the device and then withdrawn such that the filler fluid is drawn into the device from the ports.

19. The method according to any of claims 1 to 18, wherein the filler liquid is dodecamethylpentasiloxane, decane or dodecane.

20. The method according to any of claims 1 to 19, wherein the filler liquid contains a surfactant.

21. The method according to claim 20, wherein the surfactant is a sorbitan ester.