Protein binding assays

Abstract

Provided herein are methods, and compositions for the analysis of protein binding. The methods are applicable to synthesis of proteins on a microfluidic device and assays using the expressed proteins.

Description

[0001] PROTEIN BINDING ASSAYS

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods and compositions for the on-device expression and binding assays on the expressed proteins. The methods are applicable to performing protein binding assays on a microfluidic device.

[0004] BACKGROUND TO THE INVENTION

[0005] The invention relates to methods for expression of proteins on a microfluidic device, particularly cell-free protein synthesis. Cell-free protein synthesis (CFPS) regimes are attractive alternatives to cell-based expression systems as they can be treated as reagents rather than organisms, making them amenable to in-vitro experimentation techniques. Additionally, cell-free systems are less sensitive to toxic protein synthesis; are open systems that can be modulated via addition of elements due to the lack of a cell membrane; are adaptable to high-throughput experiments; and can be used to good effect in small volumes. However, many of the cellular expression regulatory control paradigms still apply (e.g. incorrect ribosome binding motifs can lead to poor binding and poor transcription; incorrect codon usage can lead to inefficient translation etc).

[0006] Efficient protein synthesis relies on having the correct nucleic acid expression construct in the correct conditions. Protein synthesis and purification can be improved by attaching additional amino acids to the protein of interest, for example sequences improving solubility or tags for purification. In order to efficiently screen the optimal cell-free conditions for expression of a particular protein sequences it is desirable to provide a population of nucleic acid expression constructs. Furthermore, in order to identify the best DNA construct to generate a protein of interest it is desirable to provide a population of nucleic acid expression constructs. The invention herein describes methods for the screening of nucleic acid constructs suitable for cell- free protein expression, and the use thereof.

[0007] When performing cell-free protein synthesis at microfluidic scale in a microfluidic device, such as a digital microfluidic device, it is useful to perform assays on the proteins that are synthesized from said cell-free protein synthesis reaction. However, it is difficult to perform real-time detection of proteins in a cell-free protein synthesis reaction environment. The reaction contains many other proteins and biomolecules at high concentration, making non-specific protein detection via standard protein staining methods difficult (e.g., Coomassie Brilliant Blue G-250, SYPRO™ Ruby, Silver staining). Immunostaining or affinity-based purification followed by non- specific proteins staining are equally unhelpful as significant washing on a solid support must be performed to prevent background interference. As washing is known to be difficult in microfluidic and digital microfluidic devices, background interference may become debilitating.

[0008] Currently existing luminescent complementation approaches cannot achieve prolonged realtime detection in cell-free protein synthesis reactions, which often extend beyond 6 hours. This limit is due to a combination of reasons including O2 consumption by the luminescencegenerating enzyme that competes with cell-free protein synthesis O2 requirements and temporary or permanent exhaustion of luminescent substrate over 3 - 24 hours of recombinant protein expression detection.

[0009] Proteins of interest may be expressed as a fusion to a fluorescent protein, such as green fluorescent protein (GFP). However, GFP is a 26.9 kDa protein, which is the typical size for most fluorescent proteins. Tags of this size increase the total size of the protein of interest, especially if the protein of interest must be tagged with other large fusion proteins such as maltose-binding protein (MBP), which is 42.5 kDa. Given the average size of a human protein is ~52 kDa and the average size of an E. coli protein is ~35 kDa (Kim, Y. E. et al. Annu. Rev. Biochem. 2013. 82:323- 355), the addition of a comparably sized fluorescent protein tag can significantly change the biological function and biophysical characteristics of a protein.

[0010] Many pieces of prior art disclose the use of sub-component tags for monitoring expression in a cellular system. For example US 7,666,606 discloses protein-protein interaction detection systems using microdomains.

[0011] Schinn et al. Biotechnol. Bioeng 114 10 October 2017 2412-2417.

[0012] (https: / / onlinelibrarv.wilev.com / doi / 10.1002 / bit.263Q5) discloses Rapid in-vitro screening for the location-dependent effects of unnatural amino acids on protein expression and activity - Schinn - 2017 - Biotechnology and Bioengineering - Wiley Online Library.

[0013] WO2022 / 038353 describes a method for measuring protein expression levels using split fluorescent protein systems. The level of expressed protein is measured in the presence of an excess of detector species, thereby measuring a single interaction which assembles a fluorescent protein. US20050221343 provides a protein labelling and detection system based on selfcomplementing fragments of fluorescent and chromophoric proteins, such as those derived from Aequorea victoria Green Fluorescent Protein (GFP). This system can be used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

[0014] US20100062451 provides methods and compositions for the production of biomolecules on beads or particles, as well as for the transfer of substances and compounds, such as biomolecules, from one surface to another.

[0015] US20120149128 describes a set of assay reagents and methods for analyte assays, particularly biological assays such as protein assays.

[0016] US20170131279 describes methods to identify and quantify the presence, type, and amount of reactants and products of chemical reactions, including using a digital droplet ELISA assay. This is achieved by incorporating components of a chemical reaction in a droplet and allowing the chemical reaction to occur in the droplet. The optionally immobilised reaction components can be detected and quantified by measuring the detectable label resulting from the reaction.

[0017] SUMMARY

[0018] Here we report the preparation and screening of proteins in a droplet on a digital microfluidic device and binding assays using the expressed proteins. The assays can result in assembly of a fluorescent protein, which is detected in the device. The assays may be performed to measure the binding affinity using the presence of both the sub-components of a fluorescent protein as a detector. Thus binding partner Y can be modified with for example Fn and Fuo added in order to determine the presence of Fn as assembled Fi-u is fluorescent. The interaction of X and Y-Fn and Fi-io allows formation of X-Y-Fm and the fluorescent signal generated is therefore a measure of the binding between X and Y. Described herein are such bead based binding assays performed on a digital microfluidic device.

[0019] Disclosed is a method for identifying protein binding using a fluorescent assay in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet containing a protein (X) immobilised on a solid support; b. exposing the protein (X) to potential binding partners (Y) wherein the binding partners are detectable by fluorescence or carry a detectable tag; c. removing unbound binding partners; d. optionally adding a detector species which makes the binding partners fluorescent; and e. determining the level of fluorescent signal within the droplets, thereby measuring the level of binding partner (Y) bound to the protein (X).

[0020] Binding partners X and / or Y may be expressed on the device. The expression may use cell-free protein synthesis (CFPS). The expression may be in the same droplets or in separate droplets. The droplets may be merged after expression and capture of one of the proteins to the solid supports. Any expression system can be used to express the proteins. The transcription and translation may use a mammalian, insect, plant or bacterial derived expression system. The transcription and translation may use a human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary (CHO) lysate system, a wheat germ cell-free system, a E. coli whole cell lysate system, a tobacco cell lysate system, a yeast lysate system or in a system of purified recombinant elements (PURE) or a mixture thereof. The reagents may be blended or supplemented to optimise expression.

[0021] Any solid support may be used. The solid support may be magnetic or paramagnetic beads. The beads may contain agents which bind to the proteins allowing capture and purification.

[0022] Binding partners (Y) may carry a suitable label for detection. The label may be a fluorophore. The partners may carry a detectable tag such as a binding agent or a sub-component of a fluorescent protein. The detectable tag may contain for example amino acid sequences from GFPn, sfGFPn or ccGFPn.

[0023] The detector species may bind to the tag in order to produce a signal or to capture the label. The detector species may be for example GFPi-io, sfGFPi-io or ccGFPi-io.

[0024] The binding partners can be any affinity ligands, for example a small molecule, nucleic acid or amino acid sequence. The binding partner (Y) can contain for example a single chain binding sequence such as a VHH or nanobody.

[0025] Assays may be performed in parallel on multiple species. For example a population of (n) expressed proteins may be expressed in separate droplets, a population of (m) potential binding partners expressed in separate droplets and the droplets of expressed proteins split into at least (m) number of droplets and binding partners split into at least (n) number droplets and the droplets combined to perform (n)x(m) number of binding assays simultaneously on the device. Disclosed herein is a method comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes; b. binding the expressed proteins to magnetic beads; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector, thereby measuring the presence of the binding partners on the beads.

[0026] The protein may be captured onto beads using a binding interaction. The proteins may have a purification tag selected from:

[0027] Alfa-tag (SRLEEELRRRLTE) (SEQ ID NO: 1)

[0028] Avi-tag (GLNDIFEAQKIEWHE) (SEQ. ID NO: 2)

[0029] C-tag (EPEA) (SEQ ID NO: 3)

[0030] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 4)

[0031] Dogtag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 5)

[0032] E-tag (GAPVPYPDPLEPR) (SEQ ID NO: 6)

[0033] FLAG (DYKDDDDK) (SEQ ID NO: 7)

[0034] G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 8)

[0035] HA (YPYDVPDYA) (SEQ ID NO: 9)

[0036] His (HHHHHH) (SEQ ID NO: 10)

[0037] Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 11) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 12)

[0038] Myc (EQKLISEEDL) (SEQ ID NO: 13)

[0039] NE-Tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 14)

[0040] Poly Glutamate-tag (EEEEEEE) (SEQ ID NO: 15)

[0041] Poly Arginine-tag (RRRRRRR) (SEQ ID NO: 16)

[0042] RholD4-tag (TETSQVAPA) (SEQ ID NO: 17)

[0043] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 18)

[0044] Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 19)

[0045] SH3 (STVPVAPPRRRRG) (SEQ ID NO: 20) SNAC (GSHHW) (SEQ ID NO: 21)

[0046] Snooptag (KLGDIEFIKVNK) (SEQ. ID NO: 22)

[0047] Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 23)

[0048] Softag 3 (TQDPSRVG) (SEQ ID NO: 24)

[0049] Spot-tag (PDRVRAVSHWSS) (SEQ ID NO: 25)

[0050] Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 26)

[0051] S-tag (KETAAAKFERQHMDS) (SEQ ID NO: 27)

[0052] Strep-tag (AWAHPQPGG) (SEQ ID NO: 28) (AWRHPQFGG) (SEQ ID NO: 29)

[0053] Strep-tag II (WSHPQFEK) (SEQ ID NO: 30)

[0054] T7tag (MASMTGGQQMG) (SEQ ID NO: 31)

[0055] TC-tag (EVHTNQDPLD) (SEQ ID NO: 32)

[0056] Ty-tag (CCPGCC) (SEQ ID NO: 33)

[0057] VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 34)

[0058] Xpress-tag (DLYDDDDK) (SEQ ID NO: 35).

[0059] Disclosed herein is a method comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes, wherein the expressed proteins have a tag selected from Strep-tag or Strep-tag II; b. binding the expressed proteins to magnetic beads having streptavidin or strep- tactin; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable ccGFPn tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species comprising ccGFPi-io; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector ccGFPi-n, thereby measuring the presence of the binding partners on the beads.

[0060] The step to measure binding partners may be performed on the beads. Alternatively the bound material, either as a complex with the protein X such that X-Y is eluted or via disruption of binding such that the bound material Y is released from protein X may be removed from the supports prior to the detector being added to quantify the amount of detector tag present. The detector may be exposed to the immobilised binding tag or the tag eluted from the solid support.

[0061] Any fluorescent protein may be used for detection. The fluorescent protein may be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFPi-io. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-io. The peptide tag may be CFASTn or CFASTio and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog. The peptide tag may be ccGFPn and the further polypeptide ccGFPi-io. The peptide tag may be sfGFPn and the further polypeptide sfGFPi-io.

[0062] The fluorescent protein may be GFP. The fluorescent protein may be sfGFP. The fluorescent protein may be ccGFP.

[0063] The fluorescent proteins or sub-components of the fluorescent protein may be attached to either the expressed protein (X) or the potential binding partner (Y). The expressed protein (X) may contain GFPn, sfGFPn or ccGFPn. The potential binding partner (Y) may be attached to GFPi-io, sfGFPi-io or ccGFPi-io.

[0064] The complementary ccGFP / GFPn peptide amino acid sequence could be the following:

[0065] 1. KRDHMVLLEFVTAAGITGT (SEQ ID NO: 36)

[0066] 2. KRDHMVLHEFVTAAGITGT (SEQ. ID NO: 37)

[0067] 3. KRDHMVLHESVNAAGIT(SEQ ID NO: 38)

[0068] 4. RDHMVLHEYVNAAGIT (SEQ ID NO: 39)

[0069] 5. GDAVQIQEHAVAKYFTV (SEQ ID NO: 40)

[0070] 6. GDTVQLQEHAVAKYFTV (SEQ ID NO: 41)

[0071] 7. GETIQLQEHAVAKYFTE(SEQ ID NO: 42) or a truncated version thereof. Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof. GFPn or GFPi-io can be fused to the protein of interest or binding partner through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.

[0072] The assays may be performed in a digital microfluidic device, for example an electrowetting-on- dielectric (EWoD) device. Expression can occur on or off the device. The expression may be performed in droplets on the digital microfluidic device having an array of electrodes. The device may be for example an array of active-matrix thin film transistors (AM-TFTs). The droplets may be on the device in an oil layer, which may contain a surfactant. The droplets are typically aqueous droplets in a hydrophobic oil layer. One or both of the layers may contain a surfactant. The surfactant in the oil layer may be a non-ionic surfactant. The surfactant in the oil layer may be a sorbitan ester such as Span85.

[0073] The use of the terms "in-vitro" and "cell-free" may be used interchangeably herein.

[0074] The expressed protein may be fused to multiple tags to aid purification, solubility or detection. For example the protein may be fused to multiple GFPn peptide tags and the synthesis occurs in the presence of multiple GFPi-io polypeptides. For example the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi. io polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more sfCherryi-io polypeptides and one or more GFPi-io polypeptides.

[0075] BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1: A schematic outlining the assay process where the binding species is labelled before binding. The binding interaction can be detected via the interaction of the partners.

[0077] Figure 2: A schematic outlining the assay process where the binding species is labelled using a detector. The detector can become fluorescent via assembly. The binding interaction can be detected via the interaction of the partners. The three components can be mixed in a single composition, or the binding to the immobilised protein can be performed first, followed by using the detection tag to capture the detector.

[0078] Figure 3: Fluorescence image of expression droplets post ccGFPi-io detector incubation at 242 ms exposure time.

[0079] Figure 4: Fluorescence measurements shown after expression from the DNA reservoir that the cell-free blend droplet received a droplet from. Ports A3, A5, and A7 were loaded with full workflow control (DNA encoding MBP), complementation control (recombinant MBP), and Universal control (ccGFP without a DET tag). All other ports were loaded with buffer blank. After incubation with ccGFPi-io detector protein, fluorescence was expected from A3, A5, and A7 ports. No fluorescence was expected from the other ports.

[0080] Figure 5: Fluorescence image of bead droplets at 568 ms exposure shows signal from beads incubated with ccGFPi-io detector droplets.

[0081] Figure 6: Purification concentrations. As expected the complementation control did not purify as it does not have a Strep tag to bind to the beads. It was possible to detect protein-protein interactions on beads on device. The fluorescence visible was due to the interaction of the ccGFPi-io detector protein and the immobilised MBP with a ccGFPn tag.

[0082] Figure 7: Plate reader data from fluorescent beads in solution. The data shows that proteinprotein interactions on strep beads are possible and detectable. The universal control and full workflow control had visible fluorescence. As expected the complementation control did not purify as it does not have a Strep tag.

[0083] Figure 8: Schematic assay for protein / protein interactions. A solid support has an immobilised protein of interest (POI 1). Interaction with a second protein of interest / ligand (POI 2) having a detector tag allows generation of signal specific to the interaction between POI 1 and POI 2.

[0084] Figure 9: Expression and purification of PopB and PcrH.

[0085] Figure 10: shows the results for expression of POI 1 and POI 2 (PopB / PcrH) studies on device and in tube (pM). Legend: D = DET-tag and S = Strep-tag.

[0086] Figures 11a and lib show a schematic of an on device assay according to the present invention. Figure 11a shows a co-expression assay where a first protein contains a tag for immobilisation (strep) and a second protein contains a tag for detection. A signal is only generated if the detector tag can be immobilised via a protein-protein interaction. Figure lib shows a control experiment which shows the protein can be expressed and detected by having the immobilisation tag and detector tag on the same sequence.

[0087] DETAILED DESCRIPTION OF THE INVENTION

[0088] Disclosed herein are improved protein binding / affinity assays. Disclosed is a method for determining protein binding using a fluorescent readout in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet containing an immobilised protein (X) and a potential binding partner (Y), where Y can be detected; b. allowing the expressed protein (X) and binding partner (Y) to bind; c. removing unbound binding partners from the immobilised proteins; d. optionally adding a detector species which binds to Y where Y is not already detectable; and e. determining the level of fluorescent signal within the droplets, thereby measuring the level of binding partner (Y) bound to the expressed protein (X).

[0089] Any fluorescent protein may be used. The fluorescent protein may be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFPi-io. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-io. The peptide tag may be CFASTn or CFASTio and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog. The peptide tag may be ccGFPn and the further polypeptide ccGFPi-io.

[0090] The fluorescent protein may be GFP. The fluorescent protein may be sfGFP. The fluorescent protein may be ccGFP.

[0091] The detector may be assembled and thereby the immobilised material becomes fluorescent as a result of the immobilised protein binding with the binding partner which binds to the detector. Alternatively binding partners (Y) may be fluorescently labelled. Excess unbound material may be removed by washing the immobilised material.

[0092] Exemplary assays are performed by measuring the binding affinity in a three-component system containing both the sub-components of a fluorescent protein. Thus immobilised protein species X and binding partner Y which is modified with for example Fn and detector species FMO assemble to form immobilised complex X-Y-Fm where assembled F is fluorescent. The immobilised material only becomes fluorescent if X binds to Y, hence capturing Fn. The interaction of X and Y allows formation of Fm and the fluorescent signal generated is therefore a measure of the binding between X and Y.

[0093] Disclosed is a method for the cell-free expression of peptides or proteins in a digital microfluidic device. The droplets having the components required for cell-free protein synthesis (CFPS), otherwise known as in-vitro protein synthesis, can be manipulated by electrowetting in order to effect and improve protein expression. The immobilised protein can be any protein of interest. For example the immobilised protein can be an antibody or part thereof. The immobilised protein can be a nanobody, which lacks a light chain or Fc domain and is therefore significantly smaller than a standard antibody. Nanobodies have a high degree of flexibility at their antigen-binding interface. The screening of nanobodies with libraries of potential binding partners can be carried out.

[0094] The potential binding partners (Y) can be for example small molecules, therapeutics, antigens, lipids, nucleic acid sequences or amino acid sequences. The amino acid sequences can be part of any protein of interest. For example the binding partners can be an antibody or part thereof. The binding partners can be nanobodies. The screening of nanobodies with libraries of potential binding partners can be carried out if the nanobodies are expressed with a fluorescent protein or sub-component thereof attached.

[0095] Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A / C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path.

[0096] As an alternative to microfluidic channel systems, droplets can also be generated and manipulated on planar surfaces using digital microfluidics (DMF). In contrast to channel based microfluidics, DMF utilizes alternating currents on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation.

[0097] Cell-free protein synthesis, also known as in-vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in-vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275- 284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261- 268).

[0098] To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other bioproducts adsorb and / or denature to hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Langmuir 2011, 27, 13, 8586-8594).

[0099] Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air. However, at elevated temperatures or over prolonged periods, the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops. Hence air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature suitable for enzyme activity and the duration of the synthesis needs to be prolonged for synthesized proteins levels to be detectable. Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis.

[0100] Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and a cell-free system having components for protein expression in an oil-filled environment, and moving said droplets using electrowetting. The components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.

[0101] The droplet can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0102] The droplet can be moved using any means of electrowetting. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.

[0103] The oil in the device can be any water immiscible liquid. The oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C).

[0104] A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the droplets during the protein expression. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coli cell growth (RSCAdv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air. The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0105] The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with the binding partner droplets for screening.

[0106] The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.

[0107] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in-vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.

[0108] An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process. The cell-free extract having the components for protein expression includes everything required for protein expression apart from the nucleic acid template. Thus the term includes all the relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, protein expression is initiated without further reagents being required.

[0109] Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or 'master mix' which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available. The reagents can be supplemented if desired to aid expression of particular proteins.

[0110] Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied. The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

[0111] The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the surface by electrokinetic forces by activation of particular electrodes. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. A digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage. Once the CFPS reagents have been enclosed in the droplets, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or 'power' sources, or test reagents to which it is desired to expose to the expressed protein. The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil.

[0112] The droplets can be in a bulk oil layer. A dry gaseous environment simply dries the bubbles onto the surface during the expression process, leaving comet type smears of dried material by evaporation. Thus the device is filled with liquid for the expression process. Alternatively, the aqueous droplets can be in a humidified gaseous environment. A device filled with air can be sealed and humidified in order to provide an environment that reduces evaporation of CFPS droplets.

[0113] The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.

[0114] The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of nonmovement.

[0115] Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet. The act of moving improves the level of protein expression over a droplet which remains static. Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.

[0116] The droplet can be moved using any means of electrowetting. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.

[0117] The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0118] The oil in the device can be any water immiscible or hydrophobic liquid. The oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The air in the device can be any humidified gas.

[0119] A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen from a gaseous environment. Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

[0120] Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS production methods. The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPei (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or UltraEver Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633-49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

[0121] Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011, 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing CFPS components may contain TWEEN 20 at 0.1% v / v, Triton X-100 at 0.1% v / v, and / or Pluronic F127 at 0.05% w / v.

[0122] For electrowetting on dielectrics (EWoD), the change in contact angle of reagent upon the application of electric potential is an inverse function of surface tension. Thus, for low voltage EWoD operations, reduction in surface tension is achieved by addition of surfactants to reagents, which for CFPS reactions means to the lysate and to the DNA. This results in a dilution of the lysate, and it has been seen, in experiments, that diluting or otherwise adulterating the lysate results in a decrease in expression level of the protein of interest. Thus performing CFPS on DMF where the surfactants are added to the solutions being moved will necessarily result in a dilution and adulteration of the lysate and thus a decrease in the level of protein expression. In addition to being a problem in its own right, this further complicates extrapolation of on-DMF results to in-tube predictions of protein yield. An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to 'user error,' as there is more handling of reagents. An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFPn (or similar) peptide tag, it's downstream complementation with a GFPi-io (or similar) detector polypeptide is hindered in the presence of surfactant. Removal of the surfactant from the aqueous phase is therefore advantageous.

[0123] Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil. This has the advantages of enabling CFPS reactions to proceed on-DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1% w / w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils. Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on-DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split ccGFP (e.g. ccGFPn / ccGFPi-io) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.

[0124] Affinity tags may be appended to proteins so that they can be purified from their crude biological source using an affinity technique. The purification tags may be selected from for example FLAG- tag, His-tag, GST-tag, MBP-tag, STREP-tag. The Flag® tag, also known as the DYKDDDDK-tag, is a popular protein tag that is commonly used in affinity chromatography and protein research. His tags are polyhistidine strings of amino acids, typically between 6 and 9 histidine amino acids in length.

[0125] The immobilisation or purification tag can be attached to the C or N terminus of the protein. The protein may be fused to multiple tags. The protein can be produced with a tag which enables immobilisation. The protein may be expressed to have an amino acid binding sequences. Affinity / immobilisation tag may be selected from:

[0126] Alfa-tag (SRLEEELRRRLTE)

[0127] Avi-tag (GLNDIFEAQKIEWHE) C-tag (EPEA)

[0128] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0129] Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0130] E-tag (GAPVPYPDPLEPR)

[0131] FLAG (DYKDDDDK)

[0132] G4T (EELLSKNYHLENEVARLKK)

[0133] HA (YPYDVPDYA)

[0134] His (HHHHHH)

[0135] Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0136] Myc (EQKLISEEDL)

[0137] NE-Tag (TKENPRSNQEESYDDNES)

[0138] Poly Glutamate-tag (EEEEEEE)

[0139] Poly Arginine-tag (RRRRRRR)

[0140] RholD4-tag (TETSQ.VAPA)

[0141] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQ.LRARLEHHPQ.GQ.REP)

[0142] Sdytag (DPIVMIDNDKPIT)

[0143] SH3 (STVPVAPPRRRRG)

[0144] SNAC (GSHHW)

[0145] Snooptag (KLGDIEFIKVNK)

[0146] Softag 1 (SLAELLNAGLGGS)

[0147] Softag 3 (TQ.DPSRVG)

[0148] Spot-tag (PDRVRAVSHWSS)

[0149] Spytag (AHIVMVDAYKPTK)

[0150] S-tag (KETAAAKFERQHM DS)

[0151] Strep-tag (AWAHPQ.PGG) (AWRHPQ.FGG)

[0152] Strep-tag II (WSHPQFEK)

[0153] T7tag (MASMTGGQ.Q.MG)

[0154] TC-tag (EVHTNQ.DPLD)

[0155] Ty-tag (CCPGCC)

[0156] VSV-tag (YTDIEMNRLGK)

[0157] Xpress-tag (DLYDDDDK).

[0158] The proteins may be immobilised directly onto the surface of the device. Alternatively the proteins may be immobilised onto beads or particles, for example magnetic or paramagnetic beads. The beads may contain the binding partner for the chosen tag, for example a metal ion to chelate His or streptavidin or strep-tactin to bind to the strep-tags.

[0159] The material may be bound to the beads on the device, of suitable particles already bound to proteins may be added to the device and moved to discreet locations.

[0160] Once suitable proteins are immobilised, the potential binding partner droplets are added to allow binding between partners X and Y. The immobilised partners may be washed to remove unbound Y. Where Y is directly detectable, the presence of the binding partners can be determined. Alternatively a labelling reaction using a detector species can be used. The detector species can be fluorescent to enable detection. Alternatively the binding partners can carry a detector which enables the detector species to become fluorescent such as for example a split fluorescent protein.

[0161] Binding partners can contain an amino acid sequence found in a fluorescent protein. The binding partners may carry a sequence such as GFPn, sfGFPn or ccGFPn. The ccGFP / GFPn peptide amino acid sequence could be the following:

[0162] 1. KRDHMVLLEFVTAAGITGT

[0163] 2. KRDHMVLHEFVTAAGITGT

[0164] 3. KRDHMVLHESVNAAGIT

[0165] 4. RDHMVLHEYVNAAGIT

[0166] 5. GDAVQIQEHAVAKYFTV

[0167] 6. GDTVQLQEHAVAKYFTV

[0168] 7. GETIQ.LQ.EHAVAKYFTE or a truncated version thereof. Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.

[0169] GFPn or GFPi-io can be fused to the binding partner through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.

[0170] Also disclosed are nucleic acid sequences for expressing particular tags. Nucleic acid sequences include

[0171] 5'GGTGATACCGTTCAGCTGCAAGAACATGCAGTTGCAAAATACTTTACCGTG (SEQ ID NO: 43) 5'GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA (SEQ ID NO: 44)

[0172] These sequences may be repeated one or more times to produce a protein having multiple GFPn domains.

[0173] Disclosed herein is a method comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes; b. binding the expressed proteins to magnetic beads; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector, thereby measuring the presence of the binding partners on the beads.

[0174] Disclosed herein is a method comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes, wherein the expressed proteins have a tag selected from Strep-tag or Strep-tag II; b. binding the expressed proteins to magnetic beads having streptavidin or strep-tactin; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable ccGFPn tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species comprising ccGFPi-io; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector ccGFPi-n, thereby measuring the presence of the binding partners on the beads.

[0175] A variety of binding assays can be performed. Examples of the types of assays that can be implemented include:

[0176] Multiplexed binding assays:

[0177] If for example the binding partners carry a different detection tags then the identity of the various partners can be detected. The binding partners may carry different coloured fluorophores or different tags for varying detector species. The colour the bead becomes indicates the binding as the fluorescence detected provides means of identifying the binding partner. The tag may be for example different amino acid sequences that form different coloured fluorescent proteins. Assays may measure the level of binding of two differently detectable species of partner Y. Assays may be used where a first species of Y contains a detectable tag comprising a sequence for a sub-component of first fluorescent protein and a second species of Y contains a detectable tag for a sub-component of a second fluorescent protein.

[0178] Negative binding assays:

[0179] Where a known detectable binding partner Y is present, the assay can be disrupted by the presence of other ligands that bind to the partner X. The other ligands (Y') can be unlabelled. The assay measures the absence of the detectable Y species. Where the binding of known ligand Y is seen, the presence of Y' has not displaced or inhibited the interaction of X and Y. The binding of X to Y' causes a loss of signal.

[0180] Titration assay

[0181] This technique involves serial dilution of protein X or protein Y to determine a saturating quantity of binding. The varying concentration of the reagents allows (or non-binding controls) immobilized on surface / beads and binding assessment of saturating quantity of Y. The assays may be used to determine the strength of binding for a particular ligand. The amount of protein X can be controlled by varying the amount of protein expressed, the amount of binding or the number of beads used in various droplets. An alternative assay may be for example a assay comparing binding of a parent antibody or a modified antibody. If the two antibodies are labelled with different detector species, the binding of the modified or original antibodies can be identified. In such cases the immobilised sequences X could optionally varied to study binding of the two or more variants of Y. This method identifies if the original or modified antibody is a stronger binder, and to which species of X the two sequences bind.

[0182] Devices

[0183] The manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrowetting occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal.

[0184] EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos0 - cos0o= (l / 2yLG) c.V2where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as sr. so / t, where sris dielectric constant of the insulator / dielectric, so is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant.

[0185] When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction (ref). The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator / dielectric, (t / £r)1 / 2. Thus, to reduce actuation voltage, it is required to reduce (t / £r)1 / 2(i.e., increase dielectric constant or decrease insulator / dielectric thickness). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.

[0186] High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a- Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators / dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator / dielectric devices.

[0187] Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator / dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. SU-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator / dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or SU-8; thus, the thickness of these materials is typically kept at a 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator / dielectric and enable operations with lower applied voltages. Inorganic materials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as "gate dielectrics", have been used as insulator / dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole free to avoid dielectric breakdown. Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules). One of the adverse effects of this hysteresis is reduced operational lifetime of the EWoD-based device.

[0188] Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after several operations. The required actuation voltage increases due to this charging phenomenon resulting in eventual catastrophic dielectric breakdown. The most probable explanation is that pinholes at the insulator / dielectric may allow the liquid to come into contact with the electrode causing electrolysis. Electrolysis is further facilitated by pinhole-prone or porous hydrophobic insulators.

[0189] Most of the studies to understand contact angle hysteresis on EWoD have been conducted on short time scales and with low conductivity solutions. Long duration actuations (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCI) could produce several effects other than electrolysis. The ions in solution can permeate through the hydrophobic coat (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) change in dielectric constant due to charge entrapment (which is different from interfacial charging) and (2) change in surface potential of a pH sensitive metal oxide. Both can result in reduction of electrowetting forces to manipulate aqueous droplets, leading to contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces or disables electrowetting on electrodes by inhibiting the modulation of contact angle when an electric field is applied.

[0190] An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes. The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.

[0191] The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick.

[0192] The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.

[0193] The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0194] The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0195] The electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled. The second substrate may also comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.

[0196] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.

[0197] The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on "Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing", US patent application no 2019 / 0111433, incorporated herein by reference.

[0198] Described herein are electrokinetic devices, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0199] Described herein is an electrokinetic device, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0200] The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.

[0201] "Droplet" refers to a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by carrier fluid and / or, in some instances, a gas or gaseous mixture such as ambient air. For example, a droplet may be completely surrounded by carrier fluid or may be bounded by carrier fluid and one or more surfaces of an EWoD device. Droplets may take a wide variety of shapes; non-limiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more working surface of an EWoD device. Droplets may include typical polar fluids such as water, as is the case for aqueous or non-aqueous compositions, or may be mixtures or emulsions including aqueous and non-aqueous components. Droplets may also include dispersions and suspensions, for example magnetic beads in an aqueous solvent. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes.

[0202] "Droplet operation" refers to any manipulation of one or more droplets on a microfluidic device. A droplet operation may, for example, include: loading a droplet into the DMF device; dispensing one or more droplets from a source reservoir; splitting, separating or dividing a droplet into two or more droplets; moving a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a microfluidic device; other droplet operations described herein; and / or any combination of the foregoing. The terms "merge," "merging," "combine," "combining" and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, "merging droplet A with droplet B," can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms "splitting," "separating" and "dividing" are not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e., the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term "mixing" refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of "loading" droplet operations includes but is not limited to microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and / or hydrophobic regions on surfaces and / or by physical obstacles.

[0203] Examples

[0204] Demonstration of assay by detecting immobilised detector tags

[0205] METHOD:

[0206] 4 separate solutions (full workflow control (POI having both Strep and ccGFPn tag), complementation control (POI missing strep), universal control (Strep and ccGFP, and Negative control without protein were incubated with magnetic streptactin beads for 30 minutes in RT under constant mixing using RotorFlex Tube Rotator at 40 rpm. Then, the supernatants were discarded and washed. The washed beads were incubated with a detector protein having ccGFPi-io (NDET / LW) and samples were transferred into a black 96 well plate, covered and then incubated for 5 hrs at 29°C before quantification and comparison on a fluorescent plate reader. The fluorescence of samples was measured using the plate reader, the data can be found in Figure 7. This data shows that measuring protein-protein interactions on strep beads are possible and detectable. The universal control and full workflow control had visible fluorescence. As expected the complementation control did not purify as it does not have a Strep tag.

[0207] Demonstration of assay by detecting immobilised detector tags on an electrowetting device METHOD:

[0208] The reaction was performed using the commercial Nuclera eProtein Discovery™ system according to the user manual unless indicated below:

[0209] All DNA reservoirs were loaded with buffer blank except for A3, A5, and A7, which were loaded with full workflow control (DNA encoding MBP), complementation control (recombinant MBP missing strep), and Universal control (ccGFP), respectively. Additionally, instead of elution buffer the port F10 was wash buffer (in order to not elute material from the beads). All the other ports were loaded as described in the user guide.

[0210] Protein expression was performed for 12 hours at 29°C as standard. Expression droplets were treated with a detector protein having ccGFPi-io (NDET / LW) and then incubated for 5 hrs at 29°C. Fluorescence detection is shown in Figure 3, and quantified in Figure 4. After incubation fluorescence was expected from ports A3, A5, and A7. All other ports were loaded with buffer blank. No fluorescence was expected from these droplets.

[0211] The concentration of complementation control (A5) and universal control (A7) were similar for the eight droplets measured. There were two populations for the expression results for the full workflow control (A3). Half of the droplets had expression values around 4-5 pM. While the remaining droplets were at around 6-7 pM. All of the expression droplets that received DNA from ports Al, A3, A5, and A7 were selected for purification. The beads were not captured at the final elution step. The beads in wash buffer were mixed with a detector droplet in order to label the bound material. The droplet was incubated and an FFL image was taken (Fig. 5).

[0212] As expected the complementation control did not purify as it does not have a Strep tag. There were 4 bead droplets with lower purification values than the other four. The low purification concentration droplets were purified from the lower expression concentrations droplets (Fig. 6). Thus the signal detected on the beads reflect the amount of bound material on the bead and can be used as an assay for identifying the amount of captured protein. It was possible to detect protein-protein interactions on beads on device. The fluorescence visible was due to the interaction of detector with the immobilised MBP with a DET tag.

[0213] Demonstration of protein-protein interaction using detector tags.

[0214] Protein Binding between PcrH and PopB Encoded by the Pseudomonas aeruginosa Type III Secretion System. Protein sequences

[0215] Regulatory protein PcrH

[0216] Uniprot Q.91325

[0217] MNQ.PTPSDTDQ.Q.Q.ALEAFLRDGGTLAMLRGLSEDTLEQ.LYALGFNQ.YQ.AGKWDDAQ.KIFQ.ALCMLDHY DARYFLGLGACRQ.SLGLYEQ.ALQ.SYSYGALMDINEPRFPFHAAECHLQ.LGDLDGAESGFYSARALAAAQ.PA HEALAARAGAMLEAVTARKDRAYESDNA

[0218] Translocator protein PopB

[0219] Uniprot Q.91324

[0220] MNPITLERAGLPYGVADAGDIPALGRPVARDVESLRVERLAAPAAASASGTGVALTPPSAASQQRLEVANR AEIASLVQAVGEDVGLARQVVLAGASTLLSAGLMSPQAFEIELAKITGEVENQQKKLKLTEIEQARKQNLQK MEDNQQKIRESEEAAKEAQKSGLAAKIFGWISAIASIIVGAIMVATGVGAAAGALMIAGGVMGVVSQSVQ QAAADGLISKEVMEKLGPALMGIEMAVALLAAVVSFGGSAVGGLARLGAKIGGKAAEMTASLASKVADLG GKFGSLAGQSLSHSLKLGVQVSDLTLDVANGAAQATHSGFQAKAANRQADVQESRADLTTLQGVIERLKEE

[0221] LSRMLEAFQEIM ERIFAMLQAKGETLHNLSSRPAAI

[0222] Each protein was prepared as a nucleic acid sequence and appended with a detector tag (ccGFPn) (D), a capture tag (Strep) (S) or both (DS). The sequences were expressed using a reconstituted cell-free expression system to produce the proteins attached to the relevant tags. Each expression system contains one or two proteins in order to ensure both S and D are present to enable purification and detection. The 6 expression combinations are shown in Figure 9:

[0223] PopB-DS

[0224] PopB-S & PopB-D

[0225] PopB-S & PcrH-D

[0226] PcrH-S & PopB-D

[0227] PcrH-DS

[0228] PcrH-S & PcrH-D.

[0229] The in-tube expression yield is shown in Figure 10. PcrH expresses more efficiently than PopB, therefore any binding experiments where the two proteins are co-expressed are limited by the level of expression of PopB.

[0230] Comparison of PopB expression and binding Design of the experiment is shown in Figures 11a and lib. Co-expression of PcrH-(S) (strep) (POI1) and PopB-D (detector ccGFPn) is compared with expression of PopB-(SD). The single PopB protein having both strep and det is a positive signal after purification and elution. For the coexpression system, PopB (having no strep tag) can only be obtained via a protein-protein interaction with the PcrH sequence immobilised via the strep tag on the PcrH.

[0231] Expression and purification was performed using the commercial Nuclera eProtein Discovery™ system. A expression screen using a variety of cell-free protein expression conditions is performed for 12 hours at 29 °C, after which droplets are split into pairs. Into one pair a detector protein (ccGFPi-io) is added and a complementation performed for a further 5 hours. Droplets having the highest level of expressed protein are selected for purification. The unlabelled droplets from the selected pairs are exposed to magnetic beads and a series of wash and aqueous liquid removal steps are performed. The material bound to the beads may be optionally eluted by disrupting the beading, for example using an elution buffer having biotin, and further droplets of detector added to monitor for the presence of the tag. Quantified data of the expression yield measured as the fluorescence signal from the relevant droplets is shown below:

[0232] PopB and PcrH demonstrated on-device protein-protein interactions evidenced by their purified yields when co-expressed (one Strep construct and one DET construct).

Claims

CLAIMS:

1. A method for identifying protein binding using a fluorescent assay in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet containing a protein (X) immobilised on a solid support; b. exposing the protein (X) to potential binding partners (Y) wherein the binding partners carry a detectable tag; c. removing unbound binding partners; d. adding a detector species which makes the binding partners fluorescent; and e. determining the level of fluorescent signal within the droplets, thereby measuring the level of binding partner (Y) bound to the protein (X).

2. The method according to claim 1, wherein the proteins (X) are expressed on the device and captured onto solid supports.

3. The method according to claim 1 or claim 2, wherein the binding partners (Y) are expressed on the device.

4. The method according to any one of claims 1 to 3, wherein the solid supports are magnetic beads.

5. The method according to any one of claims 1 to 4, wherein the detectable tag and detector species are components of a fluorescent protein.

6. The method according to claim 5, wherein the detectable tag contains ccGFPn.

7. The method according to any one of claims 1 to 6, wherein the detector species is ccGFPi-io.

8. The method according to any one of claims 1 to 7, wherein the binding partner (Y) is an amino acid sequence.

9. The method according to claim 8, wherein the binding partner (Y) contains a single chain binding sequence such as a VHH or nanobody.

10. The method according to claim 9, wherein the proteins (X) and binding partner (Y) are co-expressed in droplets on the digital microfluidic device having an array of electrodes.

11. The method according to claim 10, wherein the expressed protein and binding partner are co-expressed in the same droplet.

12. The method according to claim 10, wherein the expressed protein and binding partner are expressed in separate droplets on the device.

13. The method according to claim 12, wherein a population of (n) expressed proteins are expressed in separate droplets, a population of (m) potential binding partners are expressed in separate droplets and the droplets of expressed protein is split into at least (m) number of droplets and binding partners are each split into at least (n) numberdroplets and the droplets are combined to perform (n)x(m) number of binding assays simultaneously on the device.

14. A method according to any one of claims 2 to 13, wherein the protein expression occurs in human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary (CHO) lysate system, a wheat germ cell-free system, a E. coli whole cell lysate system or in a system of purified recombinant elements (PURE) or a mixture thereof.

15. The method according to any one of claims 1 to 14, wherein the droplets are in an oil layer and the oil layer contains surfactant.

16. The method according to any one of claims 1 to 15, comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes; b. binding the expressed proteins to magnetic beads; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector, thereby measuring the presence of the binding partners on the beads.

17. The method according to any one preceding claim, wherein the proteins are immobilised via a binding interaction.

18. The method according to claim 17, wherein the binding interaction uses a purification tag selected from:Alfa-tag (SRLEEELRRRLTE)Avi-tag (GLNDIFEAQKIEWHE)C-tag (EPEA)Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)Dogtag (DIPATYEFTDGKHYITNEPIPPK)E-tag (GAPVPYPDPLEPR)FLAG (DYKDDDDK)G4T (EELLSKNYHLENEVARLKK)HA (YPYDVPDYA)His (HHHHHH)Isopeptag (TDKDMTITFTNKKDAE)lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)Myc (EQKLISEEDL)NE-Tag (TKENPRSNQ.EESYDDNES)Poly Glutamate-tag (EEEEEEE)Poly Arginine-tag (RRRRRRR)RholD4-tag (TETSQ.VAPA)SBP-tag (MDEKTTGWRGGHVVEGLAGELEQ.LRARLEHHPQ.GQ.REP)Sdytag (DPIVMIDNDKPIT)SH3 (STVPVAPPRRRRG)SNAC (GSHHW)Snooptag (KLGDIEFIKVNK)Softag 1 (SLAELLNAGLGGS)Softag 3 (TQ.DPSRVG)Spot-tag (PDRVRAVSHWSS)Spytag (AHIVMVDAYKPTK)S-tag (KETAAAKFERQHMDS)Strep-tag (AWAHPQ.PGG) (AWRHPQ.FGG)Strep-tag II (WSHPQFEK)T7tag (MASMTGGQ.Q.MG)TC-tag (EVHTNQ.DPLD)Ty-tag (CCPGCC)VSV-tag (YTDIEMNRLGK)Xpress-tag (DLYDDDDK).

19. The method according to any one of claims 1 to 18, comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes, wherein the expressed proteins have a tag selected from Strep-tag or Strep-tag II; b. binding the expressed proteins to magnetic beads having streptavidin or strep- tactin; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable ccGFPn tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species comprising ccGFPi-io; g. optionally washing the beads to remove detector species; andh. measuring fluorescence from the assembled detector ccGFPi-ii, thereby measuring the presence of the binding partners on the beads.

20. The method according to any one preceding claim, wherein the assay contains a ligand which inhibits or displaces the binding of X and Y in order to determine the binding of the ligand by disrupting signal generated by the binding of X and Y.

21. The method according to any one preceding claim, wherein the assay determines the level of binding of two differently detectable species of partner Y.

22. The method according to claim 21, wherein a first species of Y contains a detectable tag comprising a sequence for a sub-component of first fluorescent protein and a second species of Y contains a detectable tag for a sub-component of a second fluorescent protein.

23. The method according to any one of claims 1 to 22, wherein the step of determining the level of fluorescent signal within the droplets is performed after the detectable tag is eluted from the solid support.

24. The method according to any one of claims 1 to 22, wherein the step of determining the level of fluorescent signal within the droplets is performed with the detectable tag remaining immobilised on the solid support.

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