Gas exchange during electrowetting operations

Abstract

Provided herein are methods for replenishing dissolved gas to aqueous droplets on a digital microfluidic device. Provided herein are methods of cell-free protein synthesis, optimized cell-free protein synthesis (CFPS) reagents, and methods for optimizing CFPS reagents to increase protein expression yield. The method is suitable for protein expression on microfluidic devices having hydrophobic surfaces by circulating a fill fluid to introduce additional oxygen.

Description

Field of the Invention

[0001] Provided herein are methods for replenishing dissolved gases to aqueous droplets on a digital microfluidic device. Provided herein are methods for reducing hypoxia in aqueous droplets on a microfluidic device. Provided herein are methods for cell-free protein synthesis, optimized cell-free protein synthesis (CFPS) conditions, and methods for optimizing CFPS to increase protein expression yields. These methods are applicable to protein expression on a microfluidic device having a hydrophobic surface and a non-aqueous filling fluid. Background of the Invention

[0003] Using droplet-based microfluidic technologies, such as digital microfluidics-based systems involving electrowetting, facilitates the handling of small volumes of reagents. However, using small reagent volumes results in rapid depletion of dissolved gases such as oxygen or carbon dioxide, which may be required for specific applications in the droplets.

[0004] Growth of cells, for example, may depend on the supply of specific gases such as, for example, carbon dioxide.

[0005] Cell-free protein synthesis (CFPS) has become an important tool for molecular biologists by playing a central role in a variety of applications. Cell-free systems can be divided into two major categories: cell extracts and reconstituted systems. Cell extracts are powerful, but complex and ill-defined 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.

[0006] The greatest advantage of CFPS is that it is the fastest way to obtain the expressed phenotype (protein) from the genotype (gene). Starting from a PCR or plasmid template, in vitro protein synthesis and functional assays can be performed within a few hours. In addition, it does not rely on host cells. However, cell extract-based systems are known to typically contain non-specific nucleases and proteases that adversely affect protein synthesis. CFPS systems are open systems that are suitable for modification by adding external components.

[0007] To date, digital microfluidic technologies, electrowetting-on-dielectric (EWoD), and electrokinetics have generally found limited use only in cell-free biology-based applications, mainly due to biofouling, in which biological components, such as proteins, nucleic acids, crude cell extracts, and other biological products, adsorb and / or denature on hydrophobic surfaces. It is well known in the art that biofouling limits the ability of EWoD devices to manipulate droplets containing biological macromolecules. Wheeler and colleagues reported that the maximum actuation time of droplets on an EWoD device containing a biological medium was 30 minutes before biofouling inhibited EWoD-based droplet actuation (Langmuir 2011, 27, 13, 8586-8594).

[0008] Protein expression generally requires an adequate supply of oxygen. The most convenient and productive way to power CFPS is via oxidative phosphorylation, where O2 is used as the final electron acceptor; however, there are other ways that involve supplementing with energy molecules that do not participate in oxidative phosphorylation. In microfluidic or digital microfluidic systems of confined droplets, there is not enough oxygen available to achieve efficient protein synthesis.

[0009] Digital microfluidic technology can be carried out in air-filled systems where droplets are manipulated on a surface in air. However, at elevated temperatures or over extended periods of time, volatile aqueous droplets simply dry out by evaporation and remain on the surface. This problem is complicated by the high surface area to volume ratio of nanoliter and microliter-sized droplets. Thus, 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 synthesis needs to be extended in order for the level of synthesized protein to be detectable.

[0010] On an EWoD device, cell lysate expression results in different outcomes. The protein expression levels in different droplets on the same device are not consistent. The inventors have realized that the dissolved gas in isolated aqueous droplets is rapidly depleted and the dissolved gas needs to be replenished. The inventors herein have employed a method of supplementing gas from the filling fluid to the droplets, improving the consistency of the CFPS system and the cell growth system on the electrowetting device.

[0011] Overview

[0012] In aqueous droplets on an electrowetting on dielectric (EWoD) device with multiple electrodes, the supply of dissolved gases such as oxygen or carbon dioxide is crucial. Due to the small volume of the liquid, oxygen may be rapidly consumed and an anoxic state may occur. Many processes, including CFPS and fluorescent protein maturation to produce a signal, require oxygen. The growth of cells requires carbon dioxide or other dissolved gases. Methods are disclosed herein for supplementing dissolved gases by circulating a base fluid within an electrowetting on dielectric (EWoD) device.

[0013] Methods are disclosed herein for maintaining or increasing anoxia by supplementing carbon dioxide to aqueous droplets.

[0014] Methods are disclosed herein for reducing anoxia in aqueous droplets in a water-immiscible filling fluid on an electrowetting on dielectric (EWoD) device with multiple electrodes. Methods are disclosed herein for introducing additional oxygen into aqueous droplets on a digital microfluidic device with multiple electrodes. The additional oxygen can be introduced by moving the filling fluid relative to the aqueous droplet and / or by moving the droplet relative to the filling fluid. The droplet can be kept stationary while the filling fluid is replenished.

[0015] Disclosed is a method for supplementing gas to an aqueous droplet in an immiscible filling fluid on an electrowetting-on-dielectric (EWoD) device with multiple electrodes, the method comprising keeping the droplet stationary and moving the filling fluid to supplement gas to the aqueous droplet, wherein a portion of the filling fluid is withdrawn from the device, thereby introducing a filling fluid with a higher level of dissolved gas.

[0016] Disclosed is a method for synthesizing proteins in an aqueous droplet in an immiscible filling fluid on an electrowetting-on-dielectric (EWoD) device with multiple electrodes, the method comprising using a reaction system having at least one template nucleic acid encoding the protein of interest and a cell-free lysate containing enzymes for protein synthesis, and moving the filling fluid to supplement oxygen to the aqueous droplet.

[0017] The filling fluid can be moved by flowing through the device. When a portion or even all of the filling fluid can be withdrawn from the device, the filling fluid can be replenished, thereby introducing a fresh filling fluid with a higher level of dissolved gas than the withdrawn fluid. Automated flow or drawing of fluid under gravity can be used. The fluid can be recycled to minimize waste of the filling fluid. The filling fluid can naturally contain dissolved gases such as oxygen. The filling fluid can be recharged by exposure to an atmosphere rich in the desired gas, for example by bubbling the gas into the filling fluid. When oxygen is used, atmospheric air will also be suitable for supplementing oxygen in the filling fluid. Thus, the filling fluid can be oxygenated or re-oxygenated, for example, by stirring or bubbling air through the oil.

[0018] Alternatively or in addition to flowing the oil, the aqueous droplet can be moved within the device, as disclosed in application WO2021 / 161048. The device can contain protein-free aqueous "dummy" droplets that can circulate within the device to mix the filling fluid. The protein-free droplets can circulate freely because there are no issues related to cross-contamination or biofouling.

[0019] Alternatively or in addition to flowing the oil and / or the dummy droplets, the aqueous droplets containing proteins can also be continuously circulated within the device. Application WO2021 / 161048 discloses the beneficial effects of droplet movement. The applicant herein has determined that circulating in a path in the form of a continuous loop is more beneficial than simple droplet movement. The aqueous protein droplets can circulate in a continuous loop through the filling fluid to mix the filling fluid and expose the droplets to different regions of the device. The device can also contain protein-free aqueous "dummy" droplets that can circulate within the device to mix the filling fluid. The protein-free droplets can circulate freely because there are no issues related to cross-contamination or biofouling.

[0020] An aqueous droplet having a protein can remain stationary while the filling fluid moves. Alternatively, the aqueous droplet can also be moved within the device.

[0021] Disclosed herein is a method for synthesizing a protein, the method comprising using a reaction system that comprises:

[0022] a. at least one template nucleic acid encoding the protein of interest; and

[0023] b. cell-free protein synthesis reagents;

[0024] wherein oxygen is introduced during the expression process to increase the protein yield.

[0025] Disclosed herein is a method for synthesizing a protein, the method comprising using a reaction system that has:

[0026] a. at least one template nucleic acid encoding the protein of interest;

[0027] b. a reagent composition containing enzymes for protein synthesis, wherein the composition is a cell lysate supplemented with oxygen and additional purified protein components.

[0028] The protein synthesis reaction reagents can be a mixture of cell lysates and purified components, such as a purified recombinant element system, i.e., protein synthesis using recombinant elements (PURE).

[0029] In particular, the enzymes for protein expression can be a mixture of cell lysates and purified enzymes.

[0030] The protein synthesis reaction reagents can comprise:

[0031] i. synthetic or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water.

[0032] The reaction system can also comprise additional components that increase the protein expression yield. The additional components can be selected from the following: cell lysates derived from naturally occurring or engineered cell lines, additional protein components, or chemical entities. The chemical entities can be one or more of polyethylene glycol, isolevuglandin, hexose, or thiogalactoside.

[0033] When synthesis is carried out on a microfluidic device, the lysate mixture can be optimized by combining the reagents on the device. For example, the reagent concentrations that produce optimal expression can be determined by blending the components in different ratios in a large number of droplets and simultaneously monitoring the expression to determine the optimal composition.

[0034] One aspect of the present invention includes an improved system for synthesizing proteins, the system having:

[0035] a. At least one template nucleic acid encoding the protein of interest; and

[0036] b. Cell-free protein synthesis reagents;

[0037] wherein the cell-free protein synthesis reagents contain synthetic or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl-5,6,7,8-tetrahydrofolic acid (FD), salts and water, and also contain one or more additional components that improve expression on an EWoD device. The additional components can be selected from polyethylene glycol, isopropyl β-D-thiogalactopyranoside, hexose, or thiogalactoside.

[0038] Cell lysates contain a mixture of proteins and other reagents obtained from cells that have not been purified or isolated for a specific protein. Cell lysates can be derived from mammalian cells, prokaryotic cells, yeast cells, plant cells, or protozoa. Cell lysates can be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa, BHK21, NS0, or Sp2 / 0 cells. Cell lysates can be derived from Escherichia coli cells, Saccharomyces cerevisiae or Pichia pastoris cells, tobacco or wheat cells, or Leishmania tarentolae.

[0039] It has been shown that using only cell lysates as reaction reagents results in variable and unreliable protein expression yields. While using only purified components as reaction reagents gives lower protein expression yields, but the results are more consistent. Combining cell lysates and PURE reagents in the reaction reagents for protein expression has shown reduced variability and more consistent protein expression yields.

[0040] In vitro transcription and translation can be coupled or uncoupled.

[0041] The expressed protein can be fused with a peptide tag. The tag can be used for purification and / or detection. The peptide tag can be a binding tag, such as poly-HIS or STREP-tag. The peptide tag can be a component of a fluorescent protein and other polypeptides, and the other polypeptides are the complementary part of the fluorescent protein. The fluorescent protein can 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 can be GFP 11 , and the other polypeptides can be GFP 1-10 . The peptide tag can be a component of sfCherry. The peptide tag can be sfCherry 11 , and the other polypeptides can be sfCherry 1-10 . The peptide tag can be CFAST 11 or CFAST 10 , and in the presence of a hydroxymethylbenzylidene rhodanine analogue, the other polypeptides can be NFAST.

[0042] For example, GFP 1-10 The polypeptide amino acid sequence can be from sfGFP:

[0043] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEK.

[0044] Alternatively, GFP 1-10 The polypeptide amino acid sequence can be further mutated from the above sequence to become brighter faster when complementary:

[0045] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK。

[0046] GFP 11 The peptide amino acid sequence should be long enough to bind to GFP 1-10 and produce a fluorescent signal. The complementary GFP 11 peptide amino acid sequence can be as follows:

[0047] KRDHMVLLEFVTAAGITGT

[0048] KRDHMVLHEFVTAAGITGT

[0049] KRDHMVLHESVNAAGIT

[0050] RDHMVLHEYVNAAGIT

[0051] GDAVQIQEHAVAKYFTV

[0052] GDTVQLQEHAVAKYFTV

[0053] GETIQLQEHAVAKYFTE

[0054] GFP 11 or GFP 1-10 can be fused to the protein of interest via an amino acid linker. In one embodiment, the oligopeptide, peptide or polypeptide linker can be 0 - 50 amino acids.

[0055] For example, sfCherry 1-10 The polypeptide amino acid sequence can be:

[0056] MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLLGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDITSHNED。

[0057] Complementary sfCherry 11 The peptide amino acid sequence can be:

[0058] YTIVEQYERAEGRHSTGG。

[0059] sfCherry 11 or sfCherry 1-10 can be fused to the protein of interest via an amino acid linker. In one embodiment, the oligopeptide, peptide or polypeptide linker can be 0 - 50 amino acids.

[0060] For example, the NFAST polypeptide amino acid sequence can be:

[0061] MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKVKVHMKKALS。

[0062] Complementary CFAST 11 The peptide amino acid sequence can be:

[0063] GDSYWVFVKRV。

[0064] Or complementary CFAST 10 The peptide amino acid sequence can be:

[0065] GDSYWVFVKR。

[0066] NFAST, CFAST 11 and / or CFAST 10 can be fused to the protein of interest via an amino acid linker. In one embodiment, the oligopeptide, peptide or polypeptide linker can be 0 - 50 amino acids.

[0067] The peptide tag can also be a component of a protein that forms a detectable substrate, such as a luminescent or chromogenic substrate. The protein can include β-galactosidase, β-lactamase, or luciferase.

[0068] The protein can be fused to multiple tags. For example, the protein can be fused to multiple GFP 11 peptide tags and synthesized in the presence of multiple GFP 1-10 polypeptides. For example, the protein can be fused to multiple sfCherry 11 peptide tags and synthesized in the presence of multiple sfCherry 1-10 polypeptides. The protein of interest can be fused to one or more sfCherry 11 peptide tags and one or more GFP 11 peptide tags and synthesized in the presence of one or more GFP 1-10 polypeptides and one or more sfCherry 1-10 polypeptides.

[0069] Any protein of interest can be synthesized. The protein can be an enzyme, such as terminal deoxynucleotidyl transferase (TdT) or a truncated form thereof, or a homologous amino acid sequence of other types of terminal deoxynucleotidyl transferase (TdT), or a homologous amino acid sequence of any type of Polμ, Polβ, Polλ, and Polθ, or a homologous amino acid sequence of any type of polymerase of family X. Brief Description of the Drawings

[0071] Figure 1 An image showing an aqueous fluid loaded into a digital microfluidic device located within an instrument. The loading of the aqueous fluid is achieved by an existing automatic injection pump, which can add or remove the filling fluid at a flow rate definable by programming. The injection pump pre-injects the filling fluid into the device and also assists in loading the aqueous reagent and the filling fluid from the loading hole by sucking the filling fluid from the opposite port.

[0072] Figure 2 A schematic diagram showing the loading process.

[0073] Figure 3 Shows the oxygen depletion (hypoxia) seen during protein expression. Images of droplets in a 144-droplet array and GFP expression in the hypoxic layer.

[0074] Figure 4 Shows the Figure 3 line scan and overlap between the reaction zone and the hypoxia distribution map based on.

[0075] Figure 5 Shows the slow recovery by simple diffusion. After 18 hours, almost no recovery of the oxygen level is seen.

[0076] Figure 6 Schematic diagrams showing the actuation of static and circulating pseudodroplets.

[0077] Figure 7 Showing the alleviation of the development of hypoxia by circulating pseudodroplets.

[0078] Figure 8 Showing hypoxia and graphical description after 18 hours.

[0079] Figure 9 Showing four cross-sections of the signal. The third row is the center of the static region. The tenth row is the center of the circulating region. The tenth row shows the most consistent intensity, with the smallest reduction due to hypoxia.

[0080] Figure 10 Showing the effects of circulating droplets of different sizes.

[0081] Figure 11 Showing Figure 10 Line scan of the image in

[0082] Figure 12 Showing the oil renewal experiment conducted at 29 °C. A complete oil renewal was performed after a total test incubation time of 3 hours (including loading, dispensing, and incubation on the device). The oil renewal was performed using a syringe pump, exchanging 2 mL of oil at a flow rate of 500 µL / min in 4 minutes using diagonal ports, while keeping all aqueous droplets in place by continuous actuation. After the oil renewal was completed, the test was allowed to reach completion without further intervention. The timing of the oil exchange was chosen to be appropriately in the middle of the exponential phase of protein expression. Figure 12 Showing the comparison with and without oil exchange.

[0083] Figure 13 Showing the analysis of data from Figure 12 The oil exchange pre-empted most of the hypoxia and led to a higher total yield and significantly lower variability between droplets.

[0084] Figure 14 Showing that renewing the oil reduces the hypoxia level. The oil renewal was performed after 5 hours of test time at 20 °C, before hypoxia appeared on the device (usually observed after 5 - 6 hours of incubation time). The test was conducted to incubate at room temperature (20 °C) until the total test time was 24 hours. In the oil renewal image, hypoxia started to appear at approximately 13 hours of incubation time, while in the baseline system it appeared at 7 hours of incubation time.

[0085] Figure 15 Showing at 24 hours of incubation following Figure 14Images. The hypoxia sensor shows a hole in the center of the device, but the fluorescent spots from protein expression are uniform, showing no signs of hypoxia. Thus, the oil renewal after 5 hours improves the fluorescent signal from the expressed protein.

[0086] Figure 16 Shows the incubation script for the two halves of the display device. The standard mixing mode is used on the right side, while the droplets move along a continuous serpentine loop pattern on the left side.

[0087] Figure 17 Shows an image of the device taken after 22 hours of test time. A single stationary droplet can be seen in the center of the device. The typical mixing mode is used on the right side of the device, forming a black hole, while the left side of the device does not show such a mode and the droplets move along it in a serpentine loop pattern. It can be seen that the fluorescent intensity of the droplets on the serpentine side appears relatively uniform compared to the right side.

[0088] Figure 18 Shows Figure 17 The heatmap. On the side of the device operating in the typical mixing mode, the average yield is 1.21 mg / mL and the CV is 47.8% (as expected to be very large due to the formation of the black hole). On the side of the device operating in a serpentine loop, the average droplet yield is 1.45 mg / mL and the CV is 6.8%. The movement of the droplets around the array seems to counteract the influence of position, especially the influence of oxygen availability. In addition to reducing the fluorescence variability between droplets, the average yield per droplet also increases on the side where there is oxygen limitation relative to the center of the device.

[0089] Figure 19a Shows a schematic diagram of the loading process (showing a single port). The device is made of a bottom plate with an electrode array (TFT) and a top plate (optionally glass, although it can be plastic) that defines the cell gap therebetween. The top glass has holes for loading reagents. The holes in the top glass are located under a plastic housing that contains access ports for loading the reagents into the cell gap. (A) Fill the device with a filling fluid to fill the cell gap and at least partially fill all access ports. (B) Use an external source, such as a multi-channel pipette (showing one channel) to place the reagent into the port. (C) The reagent sinks to the bottom end of the port, close to the access hole in the top glass. (D) Remove the filling fluid from the cell gap and introduce the reagent into the device for downstream operations.

[0090] Figure 19bA schematic diagram of the device is shown, with one port connected to a filling fluid extraction unit and multiple ports available for loading aqueous reagents. The loading process and subsequent filling fluid extraction can be driven by removing the filling fluid from one or more ports. The multiple ports can be placed on the same side or opposite sides of the device.

[0091] Oil can be taken out from two corners of the device, and the two corners can be on the same side or diagonal corners. The fluid for supplementing gas can be inhaled via the filled loading port by extracting fluid from the opposite port. A pump can be used to generate negative pressure for removal (i.e., the pump can push the oil into the microfluidic gap and fill the loading port, and then use negative pressure to pull to introduce fresh filling fluid from the loading port into the microfluidic gap). Detailed Description of the Invention

[0093] A method for supplementing gas to aqueous droplets is disclosed. The gas can be supplemented by circulating the filling fluid, either internally circulating the filling fluid or circulating the filling fluid by supplementing from an external source.

[0094] A method for growing cells is disclosed, in which hypoxic conditions are maintained by supplementing with oil containing dissolved carbon dioxide.

[0095] A method for reducing hypoxia in aqueous droplets in a water-immiscible filling fluid on an electrowetting-on-dielectric (EWoD) device with multiple electrodes is disclosed, the method including moving the filling fluid to supplement oxygen to the aqueous droplets.

[0096] A method for synthesizing proteins in aqueous droplets in a water-immiscible filling fluid on an electrowetting-on-dielectric (EWoD) device with multiple electrodes is disclosed, the method including: using a reaction system having at least one template nucleic acid encoding the protein of interest and a cell-free lysate containing enzymes for protein synthesis, and moving the filling fluid to supplement oxygen to the aqueous droplets.

[0097] A method for synthesizing proteins is disclosed herein, which includes using a reaction system, the reaction system comprising:

[0098] a. At least one template nucleic acid encoding the protein of interest; and

[0099] b. Protein synthesis reaction reagents;

[0100] wherein the oxygen present in b. has been increased to improve the yield of protein expression.

[0101] A method for synthesizing proteins in a digital microfluidic device is disclosed. Droplets having the components required for cell-free protein synthesis (CFPS), also known as in vitro protein synthesis, can be manipulated by electrokinetics to achieve and improve protein expression.

[0102] Protein expression depends on the conditions and reagents used for expression. The optimal expression system for a given protein of interest is unpredictable, and a large number of similar conditions may need to be screened to determine the optimal expression system. The EWoD device can be used to screen a large number of closely related conditions in parallel in the droplets on the device. The droplets can be blended on the device to prepare the reagents. For example, single cell lysates or reconstituted protein mixtures can be supplemented with a range of concentrations of various additional components. For example, salt screening, buffer screening, or pH screening can be performed under a range of conditions and at different concentrations.

[0103] Disclosed herein is a method for synthesizing a protein on a dielectrophoretic electro-wetting (EWoD) device, the method comprising employing a reaction system having at least one template nucleic acid encoding the protein of interest, blending droplets to form a series of droplets having different cell-free reagent compositions, the compositions comprising enzymes and template nucleic acids for protein synthesis, and monitoring the synthesis of the protein of interest in the various compositions to determine the composition suitable for expressing the protein of interest.

[0104] Electro-wetting is the alteration of the wetting properties of a surface (which is generally hydrophobic) by an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electro-wetting have been widely 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 a cartridge wall or a microfluidic tube. Embedded in the cartridge wall or the tube wall are electrodes covered by a dielectric layer, each electrode being connected to an A / C bias circuit capable of being rapidly switched on and off at regular intervals to alter the electro-wetting field characteristics of the layer. This results in the ability to direct the droplets along a given path.

[0105] As an alternative to the microfluidic channel system, digital microfluidic technology (DMF) can also be used to generate and manipulate droplets on a plane. Compared with channel-based microfluidic technology, DMF utilizes an alternating-polarity voltage signal on an electrode array to move fluids on the surface of the array. Thus, liquids can be moved on an open device by electro-wetting. Digital microfluidic technology enables precise control of the movement of droplets, including droplet fusion and separation.

[0106] Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the generation of peptides or proteins using biological machinery in a cell-free system, i.e., without using live cells. The in vitro protein synthesis environment is not limited within a cell wall and is not restricted by the conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, typically plasmid DNA or RNA from in vitro transcription. CFPS has been known for decades and many commercial systems are available. Cell-free protein synthesis includes systems based on crude lysates (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12):a023853) and systems based on recombinant, purified molecular reagents such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires high concentrations of biomacromolecules including DNA, RNA, proteins, polysaccharides, molecular crowding agents, etc. (Febs Letters 2013, 2, 58, 261-268).

[0107] Protein expression using cell lysates generally requires an adequate supply of oxygen. The most convenient and productive way to power CFPS is via oxidative phosphorylation where O2 is used as the final electron acceptor; however, there are other ways that involve supplementation with energy molecules not involved in oxidative phosphorylation. In microfluidic or digital microfluidic systems of confined droplets, there is not enough oxygen available to achieve efficient protein synthesis. The level of oxygen can be increased by stirring / mixing the filling base fluid.

[0108] Described herein are improved methods for cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for cell-free expression of peptides or proteins in a microfluidic device, wherein the method includes 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 the oil. The components for the cell-free protein synthesis droplets can be premixed before introduction into the digital microfluidic device or mixed on the digital microfluidic device.

[0109] Mixing of the filling fluid can be performed using dummy droplets. Dummy droplets generally do not contain proteins or other biomolecules. The size of the dummy droplets can be larger than the droplets in which proteins are expressed. While proteins are being expressed in adjacent droplets, the dummy droplets can be repeatedly moved for a period of at least 30 minutes. While proteins are being expressed, the droplets can be repeatedly moved for a period of at least two hours. While proteins are being expressed, the droplets can be repeatedly moved for a period of at least twelve hours. The act of moving the droplets enables oxygen to be supplied to adjacent droplets and dispersed throughout all the droplets on the device. The act of moving improves the level of protein expression compared to droplets that remain stationary.

[0110] Optionally or additionally, the protein droplets can be moved within the device. The dummy droplets and / or the protein droplets can be circulated in a continuous loop. The movement of the droplets in the path through the oil increases the oxygen available to the droplets.

[0111] To further enhance the mixing of the oil, the aspect ratio of the droplets can be changed to increase the exposed surface area for displacing the filling fluid. Thus, the droplets can be made rectangular and moved with the longer side as the leading edge to increase the displacement of the filling fluid, rather than moving square droplets.

[0112] Any electrokinetic means can be used to move the droplets. Dielectrophoretic electro-wetting on dielectric (EWoD) can be used to move the droplets. The electrical signals on the EWoD or optical EWoD devices can be delivered through segmented electrodes, active matrix thin film transistors or digital micromirrors.

[0113] The filling liquid can be a hydrophobic or non-ionic liquid. For example, the filling liquid can be decane or dodecane. The filling fluid can be a silicone oil such as dodecamethylpentasiloxane (DMPS). The filling liquid can contain a surfactant such as sorbitan esters, such as Span 85.

[0114] The non-aqueous filling 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. Before or during the expression process, the oil can be oxygenated. Optionally, the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved to position and immobilized in an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing a mounted reagent reservoir. Additionally, humidification can be achieved by connecting an aqueous reservoir to the enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration to provide a specific relative humidity (e.g., a saturated potassium sulfate solution at 30 °C).

[0115] A source of supplementary oxygen can be supplied to the droplets. For example, during the protein expression process, droplets or bubbles containing gaseous or dissolved oxygen can be located near or merged with the droplets. Additionally, a source of supplementary oxygen can be obtained by oxygenating the oil used as the filling medium. It is well known in the art that oils such as hexadecane, HFE-7500 and others can be oxygenated to support the oxygen demand for cell growth, especially for Escherichia coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by inflating the oil with pure oxygen or atmospheric air.

[0116] Droplets can be formed before entering the microfluidic device and flow into the device. Alternatively, droplets can be merged on the device. A method is included that includes merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free extract having components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0117] Droplets can be split on the device before or after expression. A method is included herein that further includes splitting an aqueous droplet into multiple droplets. If desired, the split droplets can be screened with other additives. A method is included in which one or more split droplets are merged with additive droplets for screening.

[0118] Cell-free expression of peptides or proteins can use a cell lysate with reagents for protein expression. Common components of the cell-free reaction include an energy source, an amino acid supply source, cofactors such as magnesium, and associated enzymes. The cell extract is obtained by lysing the cells of interest and removing cell walls, DNA genomes, and other debris by centrifugation. What remains is the cell machinery, including ribosomes, aminoacyl-tRNA synthetases, translation initiation factors 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 expression machinery from the cell source.

[0119] To optimize expression, the expression system can be supplemented with additional components, including purified enzymes. The additional components can include salts, cofactors, buffers, surfactants, molecular chaperones, or other protein components. The additional protein components can be selected from, for example, molecular chaperones, glycosylating enzymes, proteases, redox-active enzymes, phosphorylases, and kinases.

[0120] Expression compositions can be assembled on the device by mixing various droplets for parallel screening of various compositions.

[0121] For example, screening reagents can include:

[0122] A mixture of molecular chaperones (e.g., PUREfrex GroE mixture)

[0123] Kinase 1 (e.g., NEB CK2)

[0124] Kinase 2 (e.g., NEB PKA)

[0125] Protease 1 (e.g., NEB TEV)

[0126] Protease 2 (e.g., Merck HRV 3C)

[0127] A common metal ion mixture

[0128] Common cofactor mixtures.

[0129] The user can blend the compositions and monitor the expression level of the protein of interest under each blending condition.

[0130] Any specific nucleic acid template can be expressed using the systems described herein. Three types of nucleic acid templates for CFPS include plasmids, linear expression templates (LETs), and mRNAs. Plasmids are circular templates that can be produced in cells or synthesized. LETs can be prepared via PCR. Although the preparation of LETs is easier and faster, plasmid yields are generally higher in CFPS. mRNAs can be produced using in vitro transcription systems. The method uses a single nucleic acid template per droplet. The method can use multiple droplets each having a different nucleic acid template.

[0131] Energy sources are an important part of cell-free reactions. Typically, a separate mixture containing the required energy source, along with the supply of amino acids, is added to the extract used for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and phosphocreatine. During the expression process, the energy source can be supplemented by adding additional reagents to the droplets during the process.

[0132] Thus, the cell lysate can be supplemented with additional reagents before the template is added. Cell-free extracts with the components for protein expression will generally be manufactured as a bulk reagent or'master mix' that can be formulated into many identical droplets before different templates are added separately to different droplets. Common cell extracts currently in use are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE), and Kluyveromyces yeast (D2P system). All of these extracts are commercially available.

[0133] The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any device that merely has droplets in an oil flow in a channel. By activating specific electrodes, droplets move electrokinetically on the surface. After electrode activation, the dielectric layer becomes less hydrophobic, causing the droplets to spread on the surface. The setup of digital microfluidic (DMF) devices is known in the art and depends on the substrate used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layer, and the applied voltage.

[0134] Once the CFPS reagents have been encapsulated in droplets, additional reagents can be provided by merging the original droplets with a second droplet. The second droplet can carry any desired additional reagent, including, for example, an oxygen or 'power' source, or a test reagent that is desired to be exposed to the expressed protein.

[0135] 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, provided that the droplets do not dissolve into the bulk oil.

[0136] Thus, prior to the addition of the nucleic acid template to the droplets, the droplets containing the cell-free extract with the components for protein expression will generally be in an oil-filled environment. The template can be added by merging droplets on a microfluidic device. Alternatively, the template can be added to 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 raising the temperature. The expression system generally operates optimally at a temperature above standard room temperature, such as at 29 °C or above.

[0137] The expression process generally takes several hours. Thus, the process should be maintained for at least 30 minutes or 1 hour, generally at least 2 hours. The expression can be maintained for at least 12 hours. During the expression process, the filling fluid can be moved within the device. The movement improves the process by mixing the reagents and ensuring that there is sufficient oxygen within the droplets. The movement can be continuous or can be repeated with intermediate periods of non-movement.

[0138] Thus, while the protein is being expressed, the filling fluid can be repeatedly or continuously moved for a period of at least 30 minutes or 1 hour. While the protein is being expressed, the filling fluid can be repeatedly or continuously moved for a period of at least two hours. While the protein is being expressed, the filling fluid can be repeatedly moved for a period of at least 12 hours. The act of moving the filling fluid effects mixing within the droplets and effects the supply of oxygen or other reagents to the droplets. The act of moving improves the level of protein expression compared to a system that remains stationary.

[0139] Digital microfluidic technology (DMF) refers to a two-dimensional planar platform for a lab-on-a-chip system for microdroplet-based manipulation. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform having a set of insulating electrodes. Digital microfluidic technology can be used with analytical processes such as mass spectrometry, colorimetry, electrochemistry, and electrochemiluminescence.

[0140] The droplets can be moved using any means of electrokinetic motion. Aqueous droplets can be moved by electrowetting-on-dielectric (EWoD). Electrowetting-on-dielectric (EWoD) is a variant based on the electrowetting phenomenon of dielectric materials. During the EWoD process, a droplet of a conductive liquid is placed on a dielectric layer having insulating and hydrophobic properties. After activation of the electrodes, the dielectric layer becomes less hydrophobic, causing the droplet to spread over the surface.

[0141] Electrical signals on EWoD or optically activated amorphous silicon (a-Si) EWoD devices can be delivered through segmented electrodes, active matrix thin film transistors, or digital micromirrors. Optically activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0142] A source of supplementary oxygen can be supplied to the droplets. For example, during protein expression, droplets or bubbles containing gaseous or dissolved oxygen can merge with aqueous droplets. Alternatively, the source of oxygen can be a molecular source that releases oxygen. Alternatively, the droplets can be moved to the gas / liquid boundary to achieve an increase in oxygen diffusing from the gaseous environment. Alternatively, the oil can be oxygenated. Alternatively, the droplets can be present in a device filled with humidified air.

[0143] The droplets can be formed before entering the microfluidic device and flow into the device. Alternatively, the droplets can merge on the device. A method is included that includes merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free system having components for protein expression to form a droplet.

[0144] The droplets can be divided on the device before, during, or after expression. A method is included herein that further includes dividing the droplets into multiple droplets. Optionally, the divided droplets can be screened with other additives. A method is included where one or more of the divided droplets are merged with additive droplets for screening.

[0145] Proteins expressed by CFPS can be immobilized on solid support affinity resins through affinity tags, such as FLAG-tags, HIS-tags, GST-tags, MBP-tags, STREP-tags, or other forms of affinity tags. Optionally, fresh batches of CFPS reagents can be delivered on the resin. Thus, updated reagents can be used to perform protein synthesis, closely mimicking the industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS manufacturing methods.

[0146] Droplets can be actuated on hydrophobic surfaces of digital microfluidic devices (ACS Nano 2018, 12, 6, 6050 - 6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPel (Cytonix LLC). The hydrophobic surface can be modified in such a way as to reduce biofouling, especially biofouling due to exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface can also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Compared with general fluorocarbon-based hydrophobic surfaces, superhydrophobic surfaces prevent biofouling. Thus, superhydrophobic surfaces extend the ability 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 smooth liquid-infused porous surface (SLIPS), which can be formed by infusing a porous PTFE membrane with Krtox-103 oil (DuPont) (Lab Chip, 2019, 19, 2275).

[0147] Droplets can also contain additives to reduce the effect of biofouling on the digital microfluidic surface. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effect of biofouling on the hydrophobic or superhydrophobic surface of the digital microfluidic device (Langmuir 2011, 27, 13, 8586 - 8594). Such droplets can use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing CFPS components can contain 0.1% volume / volume of TWEEN20, 0.1% volume / volume of Triton X-100, and / or 0.05% weight / volume of Pluronic F127.

[0148] For electrowetting-on-dielectric (EWoD), the change in the reagent contact angle upon application of an electric potential is an inverse function of the surface tension. Thus, for low-voltage EWoD operation, the reduction of the surface tension is achieved by adding a surfactant to the reagent, which for CFPS reactions means adding to the lysate and adding to the DNA. This results in dilution of the lysate, and as has been seen in experiments, diluting or otherwise doping the lysate results in a decrease in the expression level of the protein of interest. Thus, performing CFPS on DMF with surfactant added to the solution being moved will necessarily result in dilution and doping of the lysate and thus a decrease in the protein expression level. In addition to being a problem in its own right, this further complicates the extrapolation of results on DMF to predictions of protein yields in tubes. Another disadvantage of having to add surfactant to the sample is that it increases the time required for sample preparation and also increases the likelihood of inconsistent results due to 'user error' as there is more reagent handling. Another disadvantage of having to add surfactant to the sample is that some downstream operations are hampered. For example, if the protein of interest is expressed in a cell-free system with a GFP 11 (or similar) peptide tag, its downstream complementation with a GFP 1-10 (or similar) detection polypeptide is hampered in the presence of the surfactant. Thus, it is advantageous to remove the surfactant from the aqueous phase.

[0149] Rather than adding surfactant to the aqueous sample, surfactant can be added to the oil, such as sorbitan esters, such as Span85 (e.g., sorbitan trioleate, Sigma Aldrich, SKU8401240025). This has the advantage of enabling the CFPS reaction to be carried out on DMF without dilution or doping. Additionally, it simplifies the sample preparation process for setting up the reaction, improving ease of use and consistency of results. Using 1% weight / weight Span85 in dodecane enables undiluted CFPS reactions on DMF and undiluted detection of the expressed non-fluorescent protein. Other surfactants besides Span85 and oils other than dodecane can be used. A range of concentrations of Span85 can be used. The surfactant can be non-ionic, anionic, cationic, amphoteric or a mixture thereof. The oil can be a mineral oil or a synthetic oil, including silicone oils, petroleum and perfluorinated oils or blends thereof. The surfactant can have an adverse effect on (1) the CFPS reaction and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and a detectant). For example, by using an oil-surfactant mixture for CFPS reactions on DMF, detection of the expressed protein can also be carried out without dilution and without adding an aqueous surfactant. It has been shown that the surfactant reduces some detection systems, including but not limited to separate GFP (e.g., GFP 11 / GFP1-10 ), the efficiency of the system, so removing the surfactant from the reagent mixture and instead adding it to the oil would be beneficial.

[0150] The peptide tag can be attached to the C-terminus or N-terminus of the protein. The peptide tag can be a component of green fluorescent protein (GFP). For example, the peptide tag can be GFP 11 , and the additional polypeptide can be GFP 1-10 . The peptide tag can be a component of sfCherry. The peptide tag can be sfCherry 11 , and the additional polypeptide can be sfCherry 1-10 .

[0151] The protein can be fused with multiple tags. For example, the protein can be fused with multiple GFP 11 peptide tags, and synthesis occurs in the presence of multiple GFP 1-10 polypeptides. For example, the protein can be fused with multiple sfCherry 11 peptide tags, and synthesis occurs in the presence of multiple sfCherry 1-10 polypeptides. The protein of interest can be fused with one or more sfCherry 11 peptide tags and one or more GFP 11 peptide tags, and synthesis occurs in the presence of one or more GFP 1-10 polypeptides and one or more sfCherry 1-10 polypeptides.

[0152] Device

[0153] The manipulation of droplets by applying an electric potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. The droplet manipulation caused by the applied electric potential is called electrowetting. Electromotive movement occurs due to a non-uniform electric field (dielectrophoresis or DEP) that affects the hydrostatic equilibrium of a dielectric liquid or a change in the contact angle of the liquid on a solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to generate a force on polarizable particles to induce their movement. The electrical signal can be transmitted to discrete electrodes, transistors, transistor arrays, or semiconductor diaphragms, the electrical properties of which can be modulated by an optical signal.

[0154] When a droplet is actuated between two electrodes covered with a hydrophobic insulator or dielectric, the EWoD phenomenon occurs. The electric field at the electrode - electrolyte interface induces a change in surface tension, which causes droplet movement due to a change in the droplet contact angle. The electrowetting effect can be quantitatively treated with the Young - Lippmann equation:

[0155]

[0156] wherein, is the contact angle when the electric field across the interface layer is zero, is the liquid-gas tension, c is the specific capacitance (given as ε r . ε0 / t, where ε r is the dielectric constant of the insulator / dielectric, ε0 is the permittivity of vacuum, and t is the thickness), and V is the applied voltage or potential. Thus, the change in the contact angle (inducing droplet movement) is a function of surface tension, electric potential, dielectric thickness, and dielectric constant.

[0157] When a droplet is actuated by EWoD, there are two sets of opposing forces acting on it: the electrowetting force induced by the electric field and the drag force, which includes the drag force and contact line friction resulting from the interaction of the droplet with the packing medium. The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all drag forces is variably determined by the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε r ). 1 / 2 Thus, in order to reduce the actuation voltage, it is necessary to reduce (t / ε r ), 1 / 2 i.e., increase the dielectric constant or reduce the insulator / dielectric thickness. To achieve low-voltage actuation, a thin insulator / dielectric layer must be used. However, the deposition of a high-quality thin insulator / dielectric layer is a technical challenge, and these thin layers are prone to damage before the desired electrowetting contact angle is large enough to drive the droplet. Therefore, most academic studies report using much higher voltages >100V on easily fabricated thick dielectric films (>3 microns) to achieve electrowetting.

[0158] However, high-voltage EWoD-based devices with thick dielectric films have limited industrial applicability, mainly because of their limited droplet multiplexing ability. The use of low-voltage devices including thin-film transistors (TFTs) and optically activated amorphous silicon layers (a-Si) has paved the way for the industrial application of EWoD-based devices because of their greater flexibility in addressing electrical signals in a highly multiplexed manner. The drive voltage of TFTs or optically activated a-Si is low (usually <15V). The bottleneck in manufacturing low-voltage devices and thus adopting low-voltage devices has been the technical challenge of depositing high-quality thin-film insulators / dielectrics. Therefore, there has been a particular need to improve the manufacturing and composition of thin-film insulator / dielectric devices.

[0159] Generally, electrodes (or array elements) for EWoD are covered by either (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings include fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of such materials as a hydrophobic coating on the dielectric is generally <100 nm and may have defects in the form of pinholes or a porous structure; thus, it is particularly important that the insulator / dielectric be free of pinholes to avoid electrical short circuits. Teflon has also been used as the insulator / dielectric, but due to its low dielectric constant and the thickness required to make it pinhole-free, it has a higher voltage requirement. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy resins (such as SU-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to the minimum contact angle hysteresis and a large contact angle with aqueous solutions, Teflon is still used as a hydrophobic outer coating on these insulator / dielectric polymers. However, there are difficulties in reliably fabricating a pinhole-free coating of parylene or SU-8 <1 micron thick; thus, the thickness of these materials generally remains at 2 - 5 microns at the cost of an increased voltage requirement for electro-wetting. It has also been reported that for repeated droplet manipulation using cell culture media, traditional EWoD devices with parylene C are prone to breakage and instability. Multilayer insulator devices deposited with metal oxides and parylene C films have been used to fabricate more robust insulators / dielectrics and can operate at a lower applied voltage. Inorganic materials such as metal oxides and semiconductor oxides, which are commonly used as "gate dielectrics" in the CMOS industry, have been used as insulators / dielectrics for EWoD devices. They offer the advantage of thin film deposition (<100 nm) using standard cleanroom methods. These materials are hydrophilic per se, require an additional hydrophobic coating, and are prone to forming pinholes due to the thin film layer deposition process. Along with the need for lower voltage operation of EWoD, recent development work has focused on (1) using materials with improved dielectric properties (e.g., using insulators / dielectrics with a high dielectric constant), (2) optimizing the manufacturing method to make the insulator / dielectric pinhole-free to avoid dielectric breakdown.

[0160] The operation of EWoD devices is affected by contact angle saturation and hysteresis, which are believed to be caused by one or a combination of the following phenomena: (1) charge trapping at the hydrophobic film or insulator / dielectric interface, (2) ion adsorption, (3) instability of the thermodynamic contact angle, (4) dielectric breakdown of the dielectric layer, (5) electrode - electrode - insulator interface capacitance (caused by the double layer effect), and (6) fouling of the surface (such as fouling of the surface by biomacromolecules). One of the adverse effects of such hysteresis is a reduction in the operating life of EWoD-based devices.

[0161] Contact angle hysteresis is thought to be the result of charge accumulation at the interface or within the hydrophobic insulator after several runs. Due to this charging phenomenon, the required driving voltage increases, leading to eventual catastrophic dielectric breakdown. The most likely explanation is that pinholes in the insulator / dielectric can allow the liquid to contact the electrode, resulting in electrolysis. Hydrophobic insulators that are prone to pinholes or porosity further facilitate electrolysis.

[0162] Most studies for understanding contact angle hysteresis on EWoD are based on short time scales and low conductivity solutions. Actuation for long durations (such as >1 hour) and high conductivity solutions (such as 1 M sodium chloride) may produce several effects other than electrolysis. Ions in the solution can permeate through the hydrophobic coating (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) a change in the dielectric constant due to charge trapping (which is different from interface charging) and (2) a change in the surface potential of pH-sensitive metal oxides. Both can lead to a decrease in the electrowetting force for manipulating aqueous droplets, resulting in contact angle hysteresis. The present inventors have previously found that the damage from high conductivity solutions reduces electrowetting on the electrodes by suppressing the modulation of the contact angle when an electric field is applied, or renders electrowetting impossible on the electrodes.

[0163] An electrokinetic device includes a first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating, the functional coating including: 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 including a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and a voltage source operably coupled to the matrix electrodes.

[0164] The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or strontium barium titanate. The thickness of the dielectric layer may be from 10 nm to 100 μm. Combinations of more than one material may be used, and the dielectric layer may include more than one sublayer, which may be made of different materials.

[0165] The conformal layer may comprise parylene, siloxane, or epoxy resin. It may be a thin protective parylene coating between the insulating dielectric and the hydrophobic coating. Generally, parylene is used as a dielectric layer on simple devices. In the present invention, the reason for depositing parylene is not to improve the insulating / dielectric properties, such as reducing pinholes, but to act as a conformal layer between the dielectric layer and the hydrophobic layer. The inventors have found that, contrary to other similar insulating coatings of the same thickness, such as PDMS (polydimethylsiloxane), parylene prevents contact angle hysteresis caused by highly conductive solutions or solutions that deviate from neutral pH for a long time. The thickness of the conformal layer may be from 10 nm to 100 μm.

[0166] The hydrophobic layer may comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide-polystyrene nanocomposite, a zinc oxide-polystyrene nanocomposite, precipitated calcium carbonate, a carbon nanotube structure, a silica nanocoating, or a smooth liquid-infused porous coating.

[0167] The element may include one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin-film semiconductor whose electrical properties can be modulated by incident light; and a thin-film photoconductor whose properties can be modulated by incident light.

[0168] The functional coating may include a dielectric layer containing silicon nitride, a conformal layer containing parylene, and a hydrophobic layer containing an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0169] The electrokinetic device may include a controller for regulating the voltage supplied to a single matrix electrode. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each thin-film transistor is coupled to a scan line and a gate line, and the plurality of gate lines are operably connected to the controller. This enables all individual elements to be controlled individually.

[0170] The second substrate may further comprise a second hydrophobic layer disposed on the second electrode. The first substrate and the second substrate may be arranged such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electrokinetic working space between the hydrophobic layers.

[0171] The method is particularly suitable for aqueous droplets having a volume of 1 μL or less.

[0172] The EWoD-based device shown and described below is an active matrix thin-film transistor device that contains a thin-film dielectric coating having a Teflon hydrophobic surface coating. These devices are based on the devices described in the patent application of E Ink Corporation entitled "Digital Microfluidic Device Comprising a Dual Substrate with Thin-Film Transistors and Capacitive Sensing", the US patent application number of which is 2019 / 0111433, which is incorporated herein by reference.

[0173] This document describes an electrokinetic device, which includes:

[0174] A first substrate having an electrode matrix, where each matrix electrode is coupled to a thin-film transistor, and where the matrix electrodes are coated with a functional coating including the following:

[0175] A dielectric layer in contact with the matrix electrodes,

[0176] A conformal layer in contact with the dielectric layer, and

[0177] A hydrophobic layer in contact with the conformal layer;

[0178] A second substrate including a top electrode;

[0179] A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0180] A voltage source operably coupled to the matrix electrodes.

[0181] This document describes an electrokinetic device, which includes:

[0182] A first substrate having an electrode matrix, where each matrix electrode is coupled to a thin-film transistor, and where the matrix electrodes are coated with a functional coating including the following:

[0183] One or more dielectric layers containing silicon nitride, hafnium oxide, or aluminum oxide in contact with the matrix electrodes,

[0184] A conformal layer containing parylene in contact with the dielectric layer, and

[0185] A hydrophobic layer in contact with the conformal layer;

[0186] A second substrate including a top electrode;

[0187] A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0188] A voltage source operably coupled to the matrix electrodes.

[0189] The electrokinetic device can be used with other components, such as, for example, devices for heating and cooling devices or kits for introducing reagents as needed.

[0190] The device can be an active matrix thin-film transistor (AM-TFT)-based device.

[0191] Also disclosed is an active matrix thin film transistor (AM-TFT) device having a substrate with a plurality of electrodes, the device including a plurality of fluid 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 an injection pump.

[0192] The device may include two substrates, at least one of which has a plurality of electrodes, and the two substrates define parallel plates that are separated by spacers to define a volume.

[0193] Fluid entry may be via holes in the upper plate or through the spacers. The inlets may be in the top substrate. The plurality of electrodes may be on the bottom substrate. The top substrate is glass or polymer and may have a thickness in the range of 0.5 mm to 20 mm.

[0194] The spacers may comprise an adhesive with beads having a defined size distribution. The spacers may comprise a polymeric material of defined thickness. The spacers may comprise glass, in which case the layers may fuse together. The spacer gap and thus the fluid height in the device may be from 50 μm to 250 μm. The spacer gap and thus the fluid height in the device may be from 100 μm to 150 μm.

[0195] The filling liquid may be moved via an automated means or may be moved under gravity. A hydrostatic head may be used to move the liquid within the device. Prior to loading the aqueous reagent, the holes are at least partially filled with a filling fluid. The filling fluid may be less dense than the aqueous phase such that the aqueous phase sinks into the holes. Alternatively, the aqueous phase may be located above the filling fluid, in which case all of the filling fluid must be withdrawn from the holes to enable the aqueous fluid to enter.

[0196] The device may be connected to a pump, such as an injection pump, a peristaltic pump, a disc pump, a diaphragm pump or a pneumatic pump. The pump enables the device to be filled with the filling liquid in an automated manner. Once filled, the pump enables partial withdrawal of the filling fluid to create a negative pressure in the device, the negative pressure sucking in the reagent and the filling fluid from the holes. Thus, the filling and withdrawal of the fluid can be carried out in an automated manner to allow for largely "non-manual" loading of the aqueous reagent. The automated filling liquid filling and withdrawal method may be integrated into an instrument that provides other functions related to the digital microfluidic device, the other functions including heating, cooling, optical, sensing, mechanical and magnetic functions.

[0197] The holes / loading ports of the device can be 90 degrees from each other. The inlets can be 180 degrees from each other. The inlets can be on 4 sides of the device. Each side can have at least 4, 8, or 12 ports. Each side can have 8 ports. The device can have 4 groups of 8 ports. The number of ports can be different on different sides of the device. For example, one side can have 8 ports while one side can have 4 ports. The device can have 8 ports on 3 sides and 16 ports on the fourth side. The ports can be offset to provide multiple rows of linear ports on one side, such as a first row and a second row, where the second row is offset behind the first row so that the source liquid can flow between the ports of the first row. The rows can be zigzag.

[0198] The spacing between the inlet ports can be 9 mm. The spacing between the inlet ports can be 4.5 mm. The spacing of the inlet ports is 4.5 mm or a multiple thereof. This will cover 24-well, 48-well, 96-well, 384-well ports. The spacing of the ports can be the same on each side of the device or can be of different sizes. In this case, the spacing refers to the distance between the centers of each inlet.

[0199] The volume of the aqueous reagent loaded into each inlet port can be from 1 μL to 50 μL. The volume can be from 1 μL to 20 μL.

[0200] The aqueous liquid can be introduced into the holes / loading ports by pipette, multi-channel pipette, syringe, blister pack, acoustic dispenser, or robotic liquid handler. The aqueous liquid can be loaded simultaneously from multiple holes, which can be on the same side or multiple sides of the device. Each hole is a separate liquid and can be the same as or different from the contents of the aqueous volume in other holes. The volume of the aqueous liquid loaded into each port can be the same or can be different.

[0201] The automatic filling and / or aspiration of the filling fluid can be controlled by software. The device can be part of a larger instrument system that provides environmental control, such as temperature control or light control, and can have analytical capabilities, such as an optical system for fluorescence or luminescence assay detection.

[0202] The position of the aqueous layer is controlled by the actuation of the electrodes to form reservoirs in defined areas. Once the aqueous liquid is drawn onto a substrate with multiple electrodes, the multiple electrodes are actuated to control the position of the aqueous liquid. Multiple reservoirs can be formed on the device.

[0203] A pump can be used to generate negative pressure for removal (i.e., the pump can push oil into the microfluidic gap and fill the loading port, and then use negative pressure to pull to introduce fresh filling fluid from the loading port into the microfluidic gap). In such an instance, the filling fluid flows through the device to the loading port and re-enters the device from the loading port to replenish the filling fluid in the microfluidic gap, where the gas dissolved in the microfluidic gap has been consumed. Example

[0204] Level of hypoxia

[0205] This example shows the O2 concentration present in the device in the presence of a 144-droplet CFPS reaction expressing green fluorescent protein. The consumption of O2 results in a hypoxic region, which affects the expression / maturation of the expressed GFP and appears as a central region with low fluorescence intensity, commonly referred to as a "black hole". The oxygen level in the device can be seen using a platinum porphyrin compound (Pt(II)OEPK(HSD)(50 μg / mL)(=HSO) in DMPS, and HSO is added to the base fluid and shows the oxygen level in the system. In the absence of O2, HSO shows fluorescence intensity, and the time-lapse photography software written for this specific purpose also provides a basis for quantifying O2 consumption and gradient formation.

[0206] Method

[0207] This experiment was designed as a layer on a standard CFPS reaction. The DMPS oil phase was supplemented with HSO, which is a 20-fold dilution of a 1 mg / mL HSD stock solution in C12 to 0.05 mg / mL in DMPS. The CFPS premix was loaded into the reservoir together with 0.05% F127 and dispensed into the HSO oil. Then, the experiment continued as a traditional CFPS reaction to Figure 8 occur in a mode of mixing overnight.

[0208] Experiment 2 allocated an expression plate of 144 droplets to promote the formation of a hypoxic black hole. The hypoxic dye was imaged using an NIR filter and a UV LED. Any weak fluorescence signal from the hypoxic dye was captured using a 5-second exposure for 24 hours. At the end of the experiment, the NIR filter was replaced with a 550 nm filter to image the final protein expression screening. Then a composite pseudo-color image can be constructed from the NIR channel and the green channel to provide a complete view of the O2 content in the device relative to protein expression. The purpose of this overlay is to verify the hypothesis that the decrease in protein expression is due to the reduction of O2, which should be indicated by the spatial overlap of the fluorescence of HSO and the decrease in protein fluorescence. The lower oxygen level in the center of the device reduces protein expression.

[0209] Results

[0210] In the absence of O2, HSO emits a fluorescent signal and is thus an indicator of hypoxia. The earliest faint signal can be detected at the marker point about 5 - 5.5 hours after entering the CFPS reaction. The dark structures seen in the image are CFPS droplets. HSD is dissolved in oil, with an excitation wavelength of 395 nm and detected through an NIR filter at 750 nm; thus it cannot be visualized simultaneously with protein expression. The droplets are actuated in a mixed mode, which causes them to be misaligned with the grid layout when they are fixed on the device.

[0211] The hypoxia signal roughly originates from the center of the device. Over time, it diffuses outward from the center of the device, which is also expected as hypoxia occurs in the region farthest from the port holes that are exposed to the ambient atmosphere and can act as a secondary source of O2. The CFPS reaction occurring at the center of the device acts as an O2 reservoir, and the process can be diffusion-limited between the O2 supply from the port holes and the consumption at the CFPS droplets.

[0212] Fluorescent images were captured using a filter replaced for GFP, and the fluorescent images were overlaid with the HSO plot to obtain a pseudo-color image with GFP and hypoxia layers ( Figure 3 ). The hypoxia zone overlaps well with the black hole, which is expected to be an important contributing factor to fluorescence inhomogeneity. The very obvious overlap of the green and red spectra indicates that the hypoxic region exists at the center of the device and expands outward with increasing time intervals.

[0213] Figure 4 : Line scan curves and overlaps between the reaction zone and the hypoxia map. The fluorescent signal originating from hypoxia overlaps with the position of the low-expression CFPS reaction zone. The formation of black holes in the 144-droplet plate is sufficiently obvious to be visually detected on the captured fluorescent image of the plate.

[0214] Figure 5 : Recovery from hypoxia after GFP-CFPS. The outer trend line is a linear curve made shortly after the CFPS reaction ended and all reaction data were acquired. The incubation phase continues on the premise that O2 will diffuse through the port holes and cause oxygen rebound, thus quenching the dye. Within the 18-hour observation window, there is some alleviation of hypoxia, which can be seen from the shrinking trend line of the HSO line scan. However, even after 18 hours, the center of the device remains hypoxic and oxygen-deficient.

[0215] Using circulating droplets to alleviate hypoxia

[0216] This experiment combined the use of dummy droplets to achieve circulation of the oil on the device by moving the oil between the passing-through and the reaction zone, bringing the O2-rich oil from the outer region of the plate towards the middle.

[0217] A 12x12 array of GFP-CFPS droplets was dispensed, followed by a 7x12 array of dummy droplets that occupied the gaps between the CFPS reaction zones. The dummy droplets were actuated to cycle around a row of CFPS reaction zones. This pattern was applied on one side of the plate, while the other half of the plate was kept in static incubation ( Figure 6 ).

[0218] The progression of hypoxia was monitored under a Basler-NIR camera, with images captured every 15 minutes for 24 hours. At the end of the 24-hour period, the plate was also imaged under a Sony DSLR camera to examine its expression. The images could then be overlaid in a pseudocolor channel to visualize both the hypoxia and expression maps simultaneously. Temporal trends could be extracted from these time-lapse images and analyzed using ImageJ ( Figure 7 ).

[0219] Hypoxia was seen to appear at around the 5-hour mark and earlier on the side with static incubation than on the side with cycling droplets. Due to the continuous movement of the droplets and the displacement of the oil on the plate, the movement of the oil also delayed the onset of hypoxia. The dummy droplets caused a distinct movement of the oil between the O2-rich edges and the O2-depleted interior of the droplet array. The lines in the hypoxia curve indicate a distinct movement of the oil between these regions, with darker regions being more O2-rich and brighter regions having lower O2 content, resulting in fluorescence of HSO. The alternating dark and bright stripes also indicate that the cyclic movement of the droplets brings oil from the edges (detected as inward-moving dark stripes) and displaces the O2-depleted oil towards the outer edges (detected as outward-moving bright stripes). Figure 8 Line scans across the static and cycling droplet sections are shown, and the reaction zones in the static incubation section show a higher signal for hypoxia relative to the dummy droplet section.

[0220] Protein expression showed a black hole effect forming at the center of the device that overlapped with the O2-depleted regions in the static part. The section with dummy droplets also showed the formation of black holes, but with a higher fluorescence intensity than the corresponding positions in the static incubation section. However, the section lacking droplets showed a fluorescence intensity closer to that of the black holes than the section with uninterrupted cycling ( Figure 9 ). The fluorescence intensity of the droplets in row 7 (cycling) was higher than that of the droplets in row 6 (static). Rows 3 and 7 can be compared, with each row equidistant from the static-cycling boundary on the plate. The fluorescence intensity of the droplets in row 7 (cycling) was significantly higher than that of the droplets in row 3 (static), indicating that the movement of the oil increased the O2 availability near the reaction zones, leading to higher protein production.

[0221] Conclusion

[0222] The loop movement pattern of the pseudo-droplets replenishes the O2 content of the small volume of oil and displaces this O2-rich oil to the O2-depleted center of the device. Higher protein yields indicate that oil displacement due to the movement of the pseudo-droplets has successfully increased O2 availability.

[0223] Testing the size adjustment of the circulating droplets

[0224] This experiment aimed to test the effect of droplet size on the previously observed bow-wave effect. The term "bow-wave effect" was used as a hypothesized approximation of the displacement of oil by the movement of aqueous droplets within the DMF device. The premise of this experiment was that if the size of the actuated droplets was larger, the displacement of oil (and thus hypoxia alleviation) would be amplified.

[0225] Methods

[0226] Droplets of the GFP-CFPS premix and circulating pseudo-droplets were dispensed from reservoirs directly opposite each other in position. At the time of dispensing, the positions of the droplets were staggered, and the movement on the plate was restricted to one dimension to avoid collisions between the pseudo-droplets and the CFPS premix. Thirty-six droplets of size 100 were dispensed and divided in half twice consecutively to form a 12x12 array of the CFPS premix. The gaps between those droplets were occupied by larger pseudo-droplets, which were dispensed as droplets of sz100 but divided only once to maintain their 7x7 size. Any larger droplets in any direction would collide into the CFPS array, so with a 12x12 CFPS array with a binding pitch of 25 px, 7 px was used as the maximum length of the droplet size. Larger droplets are only recommended for use with arrays with a larger pitch.

[0227] The path control scripted such that the GFP-CFPS premix and the pseudo-droplets were always parallel to each other in opposite directions and never on intersecting paths. The preferred path for this movement was horizontally between E2 and E1. All vertical movements were completed after dispensing but before the droplets entered the effective area of the droplet array, purely to avoid any potential collisions during droplet dispensing and positioning.

[0228] Time-lapse imaging was performed for 24 hours using time-lapse photography software, and the final protein expression was measured by fluorescence using a DSLR at an emission light of 550 nm.

[0229] Results

[0230] The movement of HSO in the device can be seen on the circulation side, i.e., bright and dark stripes. The bright stripes are HSO depleted of O2 that has been displaced outward, and the dark stripes are oil rich in O2 that has been displaced toward the anoxic center. The stripes in the sz49 experiment are significantly weaker than those in the sz25 experiment. Larger-sized droplets also displace a larger volume of oil during movement, which will help reduce the O2 gradient, and the O2 gradient is manifested by the intensity of the oil in the stripes of the displaced oil ( Figure 10 ). The anoxic curve of the plate with sz49 pseudo-droplets with circulation is significantly lower than that of the plate with sz25 pseudo-droplets, which is due to the possibly larger oil displacement ( Figure 11 ). The low anoxic signal of the circulating pseudo-droplet part from the sz49 droplets also indicates that the oil displacement occurring due to continuous movement replenishes the O2 in the central region of the plate, avoiding significant O2 depletion. Comparing the fluorescence intensities on the static side and the circulating pseudo-droplet side of the plate, observable black hole formation is seen on the static incubation side, while the other half of the plate with circulating pseudo-droplets does not show a significant decrease in fluorescence intensity between the droplets.

[0231] Conclusion

[0232] The difference in fluorescence intensity between the side with static incubation and the side with pseudo-droplets is consistent with previous experiments involving the movement of the reaction zone and the improvement of oxygen availability in the device by pseudo-droplets. Larger pseudo-droplets also show a lower anoxic signal, indicating that the effectiveness of anoxia mitigation is directly related to the oil displacement between the normoxic and anoxic zones.

[0233] Testing oil replenishment

[0234] The purpose of this experiment was to further explore the feasibility of using a dual-injection pump method of simultaneously injecting / withdrawing carrier oil to achieve oil renewal on DMF as a method of increasing available oxygen. To generate a droplet array, an LS70 lysate containing 0.05% F127 was loaded into the DMF cartridge using an injection pump. As an array of 108 droplets of size 7, no other reagents were loaded (since no DNA was added). Once the droplet array had been generated on the device, two identical Chemyx injection pumps with attached tubing were set to input oil into / withdraw oil from the cartridge at the same flow rate. The two injection pumps were set to simultaneously inject and withdraw oil, and the flow rate of the oil and the mixing mode of the droplets were varied to determine the maximum flow rate that would be tolerated before the droplets were perturbed, and to determine whether certain actuation modes (such as mixing versus stationary) had an impact on droplet stability.

[0235] Results

[0236] Set the oil flow rate to 0.1 mL / min and monitor the effect on droplet movement. At this flow rate, no effect on droplet movement was observed. The flow rate was then increased to 0.3 mL / min, 0.5 mL / min, 0.7 mL / min, and 1 mL / min. At any flow rate up to and including 1 mL / min (the highest flow rate used), no effect on droplet movement was observed. It is also worth noting that no oil overflowed from the ports and no bubble formation was observed, meaning that the inflow and outflow of the device were balanced (i.e., the volume inside the device was in a steady state). This flow rate was also much higher than the flow rates most likely to be used in single-use or continuous oil exchange experiments, so this effectively means that the flow rate will not constrain the further development of the method.

[0237] Testing oil replenishment for protein expression

[0238] The purpose of this experiment was to test oil renewal at a single time point during a trial run on the device to determine its effect on GFP expression yield and variability. It was hypothesized that replacing the oxygen-depleted oil on the device with fresh oil at some time point during the exponential protein expression phase, i.e., at a time point when hypoxia was expected to first occur but before it began to inhibit protein expression, would result in a higher total protein yield and minimized (or eliminated) black holes. This would be reflected in a higher average protein yield accompanied by a lower %CV across the entire device.

[0239] 144 droplets of GFP-CFPS premix were dispensed from the reservoir. Approximately 3 hours after the total trial time began (which included the time taken for loading, dispensing, and forming the array, approximately 1 hour 20 minutes) from the time the premix was prepared in the tube, the oil was renewed. This time was chosen based on previous experiments and corresponded to the time range when protein expression was seen to be proceeding rapidly but before any recognizable black holes appeared.

[0240] The oil renewal step was carried out at a flow rate of 0.5 mL / min, using diagonal ports for simultaneous injection and extraction. After this single time point renewal, the trial was left to incubate without further interference for a total trial time of 24 hours. The oil renewal step did not disturb any of the droplets on the device or the remaining reservoir.

[0241] Figure 12Show an image of the device at the end of the experiment. The fluorescence intensity appears uniform and no obvious black holes are seen, which is different from the control without oil renewal. The figure shows a comparison of the final images and heatmaps of the oil renewal experiment with the baseline. In the figure, the same conditional formatting rules are applied to the two protein yield heatmaps. Faint black holes can be seen on the oil renewal yield heatmap, and their shape is the same as that of the black holes appearing on the yield heatmap of the baseline run. This indicates that there is still slight hypoxia and the experiment is likely to benefit from additional oxygenation. This can be in the form of an additional oil renewal step (or a change in the timing of a single exchange) or continuous oil renewal at a flow rate sufficient to completely eliminate hypoxia. It is calculated that the average yield on the device is 1.87 mg / mL with a CV of 4.0%. In the absence of oil exchange, the average yield on the device is 1.43 mg / mL with a CV of 30.1%.

[0242] Figure 13 Show a quartile box plot of the experimental yields of the baseline ACA run relative to the ACA run with oil renewal performed. Clearly, the average yield of the runs with the oil renewal step is higher and the distribution is tighter.

[0243] HSO test

[0244] The purpose of this experiment is to test single-time-point oil renewal in the presence of hypoxia-sensing oil (HSO) at room temperature to determine the effect of oil renewal on the timing and degree of hypoxia occurrence compared to the baseline experiment without oil renewal.

[0245] 144 droplets of GFP-CFPS premix were dispensed from the reservoir. Oil renewal was performed after a total test time of 5 hours starting from the time when the premix was prepared in the tube. Based on previous experiments, this time was chosen to fall before the appearance of hypoxia on the device. The oil renewal step was carried out at an oil flow rate of 0.5 mL / min, injecting and extracting oil simultaneously using diagonal ports while all droplets were actuated in situ. A total volume of 2 mL of oil was replaced. After renewal, the experiment was placed under incubation without further interference for a total test time of 24 hours.

[0246] Figure 14 Show the time-lapse images of the cartridge at 5, 10, 13, and 15 hours. During the baseline run, hypoxia started to appear after 7 hours and was almost fully formed after 11 hours of test time. In contrast, during the oil renewal run, hypoxia started to appear only after 13 hours.

[0247] Figure 15Show the image of the device after 24 hours. Several dud droplets can be seen. At the end of the experiment, the fluorescence intensity on the device looks uniform and no obvious black hole formation is seen. Calculated, the average yield on the device is 1.66 mg / mL and the CV is 6.6%. Similar baseline runs of the standard test on ACA show a yield of 1.37 mg / mL and a CV% of 15.5%, indicating that the experiment with the oil renewal step improved the yield and had lower variability.

[0248] Analysis of the time-lapse hypoxia images shows that hypoxia on the device occurs later with oil renewal compared to the baseline (13 hours versus 5 hours of the experimental time respectively). The occurrence of hypoxia, although at a later stage, indicates that further experiments could benefit from further additional oxygenation to potentially further increase protein yield.

[0249] Loop mixing

[0250] The aim of this experiment was to run a standard test using a 144-droplet array and compare two different mixing strategies side by side. On one half of the device, a mixing mode using commands for near-continuous mixing was employed, while on the other half of the device, the full-side serpentine loop mode was run, where droplets move continuously along a circuitous path covering half of the entire array, such that each droplet spends almost equal time at each droplet position. The aim was to eliminate spatially dependent yield patterns such as black holes. Such patterns form when droplets remain in their initial positions throughout the experiment and are affected by position-dependent variables such as oxygen availability and local temperature variations.

[0251] 144 droplets of GFP-CFPS premix were dispensed from the reservoir. Then incubation was started. For the incubation phase, the device was split into two halves. Incubation on one half of the device consisted of running a typical mixing script which consisted of near-continuous mixing. On the other half of the device, a serpentine travel mode for the droplets was initiated. This mode moves all droplets along a path that criss-crosses the device. The aim of this travel mode is to average out all spatial effects on the yield, including oxygen availability, so as to equalize the yield achieved by all droplets. In the current form of the script, each loop of the script moves each droplet one position. Therefore, it takes 72 loops of the script to move a droplet through a complete path around the device. Considering the number of steps in the script and the current frame rate, it takes approximately 100 minutes for each droplet to complete the whole mode. The device was split into two halves and the serpentine mode formed by the script is shown in the following figure ( Figure 16 )

[0252] Figure 17Show the device image taken after 22 hours of test time. A single fixed droplet can be seen in the center of the device. A black hole has formed on the right hand side of the device, where a typical mixing mode is applied, while on the left hand side of the device, the droplets travel in a serpentine pattern and no such pattern is shown. It can be seen that the fluorescence intensity of the droplets on the serpentine side appears relatively uniform compared to the right hand side.

[0253] Figure 18 Show that on the side of the device operating in the typical mixing mode, the average yield is 1.21 mg / mL and the CV is 47.8% (very large as expected due to the formation of the black hole). On the side of the device where the droplets travel in a serpentine pattern, the average droplet yield is 1.45 mg / mL and the CV is 6.8%. The movement of the droplets around the array has eliminated the influence of position, especially the influence of oxygen availability. In addition to reducing the fluorescence variability between droplets, the average yield per droplet has also increased compared to the side towards the center of the device where there is oxygen limitation.

[0254] It is also worth noting that the fluorescence intensity of the serpentine droplets is on average higher than that of the droplets mixed using the typical mixing mode. However, the highest fluorescence levels observed along the edge (outer side) of the typical side of the device are higher than those observed in the serpentine droplets. This indicates that if more oxygen could be provided to the serpentine, then all droplets could reach this yield level (uniformly). That is to say, although the serpentine movement pattern helps to balance and optimize the consumption of available oxygen by the droplets, there are still inherent oxygen limitations that prevent the droplets from reaching the highest yield levels visible on the black hole side of the device.

Claims

1. A method for replenishing a gas into an aqueous droplet in a water-immiscible filling fluid on an electrowetting on dielectric (EWoD) device having a plurality of electrodes, the method comprising: Keep the droplet stationary and move the filling fluid to supply gas to the aqueous droplet, wherein a portion of the filling fluid is withdrawn from the device to introduce a filling 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, which is used for growing cells in the droplet on the device, wherein the droplet is supplemented with carbon dioxide.

4. The method according to claim 1, for synthesizing proteins in an aqueous droplet in a water-immiscible filling fluid on an electro-wetting on dielectric (EWoD) device with a plurality of electrodes, the method comprising: Adopt a reaction system, the reaction system has at least one template nucleic acid encoding the protein of interest and a cell-free lysate containing enzymes for protein synthesis, and move the filling fluid to supply oxygen to the aqueous droplet by introducing a filling fluid having a higher level of dissolved oxygen.

5. The method according to claim 1 or claim 4, wherein the aqueous droplet contains a fluorescent protein whose fluorescence intensity depends on oxygen.

6. The method according to claim 5, wherein the protein droplet remains stationary while the filling 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, NS0, 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 the following: synthetic or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, tRNA, amino acids, ribonucleoside triphosphates, 10-formyl-5,6,7,8-tetrahydrofolic acid (FD), salts, polyethylene glycol, isopropyl lactose, hexaldehyde sugar or thiogalactoside, buffers, surfactants, metal ions, molecular chaperones, cofactors or additional protein components.

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

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

16. The method according to any one of the preceding claims, 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 of the preceding claims, wherein the filling fluid is withdrawn from the device under gravity or by means of an injection pump, a peristaltic pump, a disc pump, a diaphragm pump or a pneumatic pump.

18. The method according to any one of the preceding claims, wherein the loading port on the device is filled with the filling fluid and then withdrawn such that the filling fluid is drawn into the device from the port.

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

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

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

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