Method for homogenizing concentration of beads in plurality of liquid volumes

Abstract

The invention provided herein relates to methods for normalizing the concentration of beads in droplets on microfluidic devices, as well as methods for cell-free protein synthesis and characterization of expressed proteins.

Description

TECHNICAL FIELD

[0001] Provided herein are methods for normalizing the concentration of beads in droplets on a microfluidic device, as well as methods for cell-free protein synthesis and characterization of expressed proteins.

[0002] BACKGROUND

[0003] Proteins are biological macromolecules that maintain the structural and functional integrity of cells, and many diseases are associated with malfunctioning proteins. Protein purification is an essential step for analyzing single proteins and protein complexes and identifying interactions with other proteins, DNA, or RNA. Various protein purification strategies exist to address issues of desired scale, yield, and downstream applications. However, protein production can be challenging for many reasons. One major challenge is finding a suitable expression system, for example, derived from mammalian, bacterial, fungal, or plant cells. This can take months of work.

[0004] Cell-free protein synthesis (CFPS), also known as coupled or uncoupled in vitro transcription and translation, is the production of peptides or proteins using biological machinery in a cell-free system, i.e., without using living cells. The CFPS environment is not confined within a cell wall nor limited by conditions necessary to maintain cell viability, and is capable of rapidly producing 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 crude lysate-based systems (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and reconstituted, purified molecular reagent-based systems, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires high concentrations of biological macromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, etc. (Febs Letters 2013, 2, 58, 261-268).

[0005] Split detection agents, such as the split green fluorescent protein (GFP) system are known for protein expression. A target protein with a GFP subcomponent can be detected by complementation with a detection agent with the rest of the GFP. Cabantous and Waldo describe such a system (In-vivo and in-vitro protein solubility assays using split GFP (NATURE METHODS | VOL. 3 NO. 10 | OCTOBER 2006 | 845). The system relies on expression in cells, lysis of the target protein from the cells and then exposing it to a detection agent to measure the expression level.

[0006] WO2022 / 038353 describes cell-free expression of proteins with a GFP tag in the presence of a GFP detection agent to measure the signal as expression proceeds.

[0007] Many proteins containing endogenous solubility enhancing factors are expressed. However, factors such as, for example, maltose binding protein (MBP), small ubiquitin-like modifier protein (SUMO), glutathione S-transferase (GST) or thioredoxin (TRX) have considerable size and can have undesired effects on the target protein to which they are attached. In addition, expression of large solubility elements uses a source of reagents during expression. Such sequences take time to fold before acting as solubility enhancers, so it is far from ideal to synthesize a target protein with a solubility enhancing sequence attached.

[0008] To date, protein purification and analysis generally requires complex analytical techniques, including electrophoresis or the protein to be purified. The inventors herein have developed a simplified method of protein analysis and purification which enables the characterisation of expressed proteins in crude form in a number of ways.

[0009] One of the current challenges in cell-free protein synthesis is to improve the soluble yield of expressed and purified proteins and to avoid aggregation or insolubility.

[0010] US8809068 describes a method of manipulating beads in microfluidic droplets. The method involves ensuring that the beads are evenly distributed within individual droplets but does not describe a method of ensuring that different droplets contain the same number of particles.

[0011] WO2023079310 describes a method of purifying proteins on a microfluidic device. The method involves manipulating beads within individual droplets but does not describe a method of ensuring that different droplets contain the same number of particles.

[0012] US20180243743 describes a device with a magnetic conduit for manipulating beads in microfluidic droplets. The method involves manipulating beads within individual droplets, but does not describe a method to ensure that different droplets contain the same number of particles.

[0013] WO2023 / 285821 describes an efficient method of separating and manipulating droplets on a microfluidic device.

[0014] Purification of biomolecules can be achieved using binding to beads. To compare the effectiveness of purification steps on different molecular species, the amount of beads between different volumes containing reagents needs to be uniform. Described herein is a method for improving the uniformity of bead dispersion between volumes of liquid. SUMMARY

[0016] Interfacial forces play an important role in droplet microfluidics compared to continuous flow microfluidics. The density of beads in sub-droplets generated from a reservoir is affected by: (1) the concentration and size of the beads, (2) the distribution of beads in the reservoir at the time of droplet generation, (3) variability in the size of the partitioning or splitting, and (4) migration of beads from high density regions to low density regions. Described herein is a method for improving the uniformity of bead dispersion between volumes of different liquids. Volumes of beads can be partitioned from a larger reservoir, for example to form droplets on a microfluidic device. Due to differences in partitioned volumes or differences in particle aggregation, the number of beads can differ between different droplets. The inventors herein have recognized that by repeatedly performing pairwise merging and splitting of droplets in sequence, interspersed with homogenization of the droplet collection, a uniform dispersion is achieved in which each droplet has the same concentration of suspended beads at the end of the merging and splitting process.

[0017] Described herein is a method of producing aqueous droplets with a uniform concentration of suspended beads, wherein the method comprises:

[0018] a. taking a reservoir filled with an aqueous liquid in which beads are suspended,

[0019] b. forming a plurality of droplet sub-volumes from the reservoir,

[0020] c. merging two or more droplets,

[0021] d. splitting the merged droplet, and

[0022] e. repeating steps c and d one or more times such that each finally split droplet contains a uniform concentration of suspended beads.

[0023] Described herein is a method of producing aqueous droplets with a uniform concentration of suspended beads, wherein the method comprises:

[0024] a. a reservoir containing an aqueous liquid with beads suspended therein,

[0025] b. forming a plurality of droplet subvolumes from the reservoir,

[0026] c. merging two or more droplets,

[0027] d. splitting the merged droplets,

[0028] e. rearranging the collection of droplets, and

[0029] f. repeating steps c, d, and e one or more times such that each final split droplet contains a uniform concentration of suspended beads.

[0030] The step of rearranging the droplets helps to counteract biases introduced by environmental parameters, such as non-uniform cell spacing or electro-wetting force gradients. The paired merging and splitting in successive iterations does not involve the same pair of droplets. It is critical that not only the same two droplets are repeatedly merged and split, whether by mixing between the droplets, or by alternating merging with different adjacent droplets, or a combination of both. Thus, different pairs of droplets are necessarily interacting with other pairs of droplets on the device. Each droplet formed from the reservoir has the potential to merge and split with every other droplet formed from the reservoir.

[0031] For example, four initial droplets A, B, C, D can be generated, containing different numbers of beads in the four droplets. A / B and C / D are merged to form two droplets, A+B and C+D. The two droplets are split into four droplets, A', B', C', D'. A' and B' have a lower degree of variation than A and B. C' and D' have a lower degree of variation than C and D. In a second mixing, A' / C' and B' / D' are mixed to form two droplets A' / C' and B' / D'. These two droplets are again split into four droplets A", B", C", D". The positions of A" and B" can be interchanged, so that A" is next to D" and B" is next to C". In a third merging step, A" can be mixed with D" and B" can be mixed with C". After the third split, the concentration of beads in the four final droplets should be more uniform than the starting concentrations in A, B, C, and D. The merging and splitting process can be repeated through multiple cycles to further improve uniformity.

[0032] Alternatively, the four droplets can be merged into a single droplet A+B+C+D, and then split into four droplets. The merging and splitting process can be repeated through multiple cycles to further improve uniformity among all the split droplets.

[0033] A method is disclosed that relies on parallel processing of a fluid volume containing beads. The fluid volume can be less than 1 μL. The fluid volume can be less than 100 nL.

[0034] The beads can be made of any particular material. In some embodiments, the beads can be magnetic. The beads can be non-magnetic.

[0035] Any number of droplets can be generated. For example, 8 or more droplets can be generated. For example, 16 or more droplets can be generated. For example, 32 or more droplets can be generated. For 32 droplets, each droplet can be mixed with each of the other 31 droplets by the following process: merging to form 16 droplets, splitting the 16 merged droplets, rotating a subset of the 32 droplets, and repeating.

[0036] Any concentration of beads can be used. Where high bead concentrations are used, the method is advantageous because higher concentrations are more difficult to distribute uniformly. For example, the beads can be, for example, greater than 5% of the volume of the droplet. The beads can be 5% to 30% volume / volume. The beads can be greater than 20% volume / volume. The final distribution can be about 20% v / v of homogenized concentration. The final distribution of homogenized bead concentration in each droplet can be 20% v / v + / - 10%.

[0037] The liquid volume can be a droplet on a digital microfluidic device, which can include an active matrix thin film transistor.

[0038] The beads can be any particular size, for example, greater than 1 μm in diameter. The beads can be greater than 10 μm in diameter. The beads can be greater than 25 μm in diameter. The beads can be magnetic beads of the stated size.

[0039] The beads can be used to bind to a substance in an aqueous phase, for example, to perform a purification step. Uniform droplets of beads can be merged with droplets having a substance bound to the beads. The substance can be a biomolecule, for example, a nucleic acid or a protein.

[0040] The invention can include a method for determining the degree of aggregation of a protein expressed in a cell-free system, comprising expressing a protein of interest (POI) in a droplet on a digital microfluidic device, wherein the expressed POI is measured using a detectable signal, and the levels of soluble and insoluble POI are measured. The level of aggregation can be used to determine whether the protein is expressed as soluble or insoluble. The soluble protein can be subsequently used for purification, for example, using magnetic beads. The soluble protein can be further purified, while proteins with a high degree of aggregation are less likely to be purified or have activity.

[0041] A method is disclosed for synthesizing, characterizing, and purifying one or more proteins having a detection tag and a binding tag, the method comprising the steps of:

[0042] i mixing the cell-free protein expression system with one or more nucleic acid templates to form a plurality of pooled liquid volumes;

[0043] iia splitting the pooled volumes into at least two aliquots and effecting protein expression in at least two aliquots; or

[0044] iib effecting protein expression and then splitting into at least two aliquots;

[0045] iii adding to at least one aliquot a detection reagent that binds to a detection tag to determine protein expression levels and determine conditions under which the protein is expressed in soluble form;

[0046] iv selecting a volume in which the protein has been expressed in soluble form and adding magnetic beads to a subpopulation of aliquots, wherein the beads are bound to a binding tag and the beads in the droplets have been pooled and split one or more times such that each final split droplet contains a uniform concentration of suspended beads;

[0047] v separating the magnetic beads from the expression reagents;

[0048] vi optionally adding a wash reagent to the magnetic beads to remove unbound material;

[0049] vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and

[0050] viii adding to at least one eluted aliquot a detection reagent that binds to a detection tag to determine protein purification levels and determine conditions under which purified soluble protein is obtained.

[0051] The extent of aggregation can be measured directly, for example using light scattering, or can be viewed using a visual marker. The detection can be viewed in the form of optically detectable aggregates that cause light scattering, or the detection can produce aggregates that can be detected by a label, for example using a fluorescent label. Light scattering can be measured at an angle different from the incident light. The POI can have a binding sequence that binds to a detection moiety. The detection moiety can be added after expression by combining additional droplets.

[0052] The extent of aggregation can be measured by the number of aggregates, the area of aggregates, the intensity count of aggregates, or by using a measure of dispersity.

[0053] The POI can be expressed with a binding sequence that binds to the detection moiety. In this case, additional droplets containing the detection moiety can be added to the droplets containing the POI to produce a detectable signal in the combined droplets. The binding sequence can contain four or more amino acids. The binding sequence can contain 4-30 amino acids.

[0054] The detection moiety can be a protein. The detection moiety can comprise a component of a fluorescent protein, such as sfGFP or ccGFP.

[0055] The expressed protein can comprise a sequence that acts as a solubility enhancer, such as selected from the group consisting of:

[0056]

[0057]

[0058] The detection tag can be one component of a fluorescent protein and the detection agent can be a complementary portion 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, miRFP670 nano. For example, the tag can be GFP 11 and the detection agent can be GFP 1-10 . The tag can be one component of sfCherry. The tag can be sfCherry 11 and the detection agent can be sfCherry 1-10 . In the presence of a hydroxybenzylidene rhodanine analogue, the tag can be CFAST 11 or CFAST 10 and the detection agent can be NFAST.

[0059] The tag can be ccGFP 11 and the detection agent can be ccGFP 1-10 .

[0060] For example, the GFP 1-10 polypeptide amino acid sequence can be derived from sfGFP:

[0061] SEQ ID NO: 1

[0062] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEK

[0063] GFP 1-10 The polypeptide amino acid sequence can be further mutated from the above sequence to become brighter more quickly after complementation. The sequence can have greater than 90% homology to any of the sequences mentioned herein. The sequence can have greater than 95% homology to any of the sequences mentioned herein.

[0064] SEQ ID NO: 2

[0065] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK

[0066] GFP 1-10 The polypeptide amino acid sequence can also be derived from ccGFP, having greater than 90% or greater than 95% homology to the following sequence:

[0067] SEQ ID NO: 3 (ccGFP 1-11 )

[0068] MSLSKQVVKEDMKMTYHMDGCVNGHYFTIEGEGTGKPFKGQKTLKLRVTEGGPLPFAFDILSATFTYGNRCFCDYPEDMPDYFKQSLPEGYSWERTMMYEDGACGTASAHISLDKNGFVHNSTFHGVNFPANGPVMKKKGVNWEPSSEKITACDGILKGDVTMFLVLEGGHRLKCLFQTTYKADKVVKMPPNHIIEHRLVRSEDGDAVQIQEHAVAKYFTV

[0069] SEQ ID NO: 4 (ccGFP 1-10 )

[0070] MSLSKQVVKEDMKMTYHMDGCVNGHYFTIEGEGTGKPFKGQKTLKLRVTEGGPLPFAFDILSATFTYGNRCFCDYPEDMPDYFKQSLPEGYSWERTMMYEDGACGTASAHISLDKNGFVHNSTFHGVNFPANGPVMKKKGVNWEPSSEKITACDGILKGDVTMFLVLEGGHRLKCLFQTTYKADKVVKMPPNHIIEHRLVRSED

[0071] SEQ ID NO 5 (ccGFP 1-10 )

[0072] MSMEKQVLKENMKTTYHMDGSVDGHYFEIEGEGTGNPFKGEQELKLRVTKGGPLPFAFDILSPTFTYGNRVFTDYPEDMPDYFKQSLPEGYSWERTMMYEDGATATASARISLDKNGFVHKSTFHGENFPANGPVMKKKGVDWEPSSETITPEDGILKGDVEMFLVLEGGQRLKALFQTTYKANKVVKMPPRHKIEHRLVRS

[0073] Nucleic acid sequence expressing SEQ ID NO 5 ccGFP 1-10 ; SEQ ID NO: 6

[0074] 5' atgagcatggaaaaacaggtgctgaaagaaaacatgaaaaccacctatcacatggatggtagcgttgatggtcactattttgaaattgaaggtgaaggcaccggcaatccgtttaaaggtgaacaagaactgaaactgcgtgttaccaaaggtggtccgctgccgtttgcatttgatattctgagcccgacctttacctatggtaatcgtgtttttaccgactatccggaagatatgccggattatttcaaacagagcctgccggaaggttatagctgggaacgtaccatgatgtatgaagatggtgcaaccgcaaccgccagcgcacgtattagcctggataaaaatggttttgtgcataagagcacctttcacggtgaaaactttccggcaaatggtccggttatgaaaaagaaaggtgttgattgggaaccgagcagcgaaaccattacaccggaagatggtattctgaaaggtgatgttgaaatgtttctggttctggaaggtggtcagcgtctgaaagccctgtttcagaccacctataaagccaataaagtggttaaaatgcctccgcgtcataaaattgaacatcgtctggttcgtagc

[0075] SEQ ID NO 7

[0076] MSMSKQVLKENMKTTYHMDGSVNGHYFTIEGEGTGNPFKGQQSLKLRVTKGGPLPFAFDILSPTFTYGNRVFTDYPEDMPDYFKQSLPEGYSWERTMMYEDGATATASARISLDKNGFVHKSTFHGENFPANGPVMKKKGVNWEPSSETITPSDGILKGDVTMFLVLEGGQRLKALFQTTYKANKVVKMPPRHKIEHRLVRS

[0077] SEQ ID NO 7 ccGFP 1-10

[0078] ​5' atgagcatgagcaaacaggtgctgaaagaaaatatgaaaaccacctatcacatggatggtagcgttaatggtcactattttaccattgaaggtgaaggcaccggtaatccgtttaaaggtcagcagagcctgaaactgcgtgttaccaaaggtggtccgctgccgtttgcatttgatattctgagcccgacctttacctatggtaatcgtgtttttaccgactatccggaagatatgccggattatttcaaacagagcctgccggaaggttatagctgggaacgtaccatgatgtatgaagatggtgcaaccgcaaccgccagcgcacgtattagcctggataaaaatggttttgtgcataagagcacctttcacggtgaaaactttccggcaaatggtccggttatgaaaaagaaaggtgttaattgggaaccgagcagcgaaaccattacaccgagtgatggtattctgaaaggtgatgttaccatgtttctggttctggaaggtggtcagcgtctgaaagccctgtttcagaccacctataaagccaataaagtggttaaaatgcctccgcgtcataaaattgaacatcgtctggttcgtagc

[0079] complementary GFP 11 The peptide amino acid sequence can be:

[0080] 1. KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9)

[0081] 2. KRDHMVLHEFVTAAGITGT (SEQ ID NO: 10)

[0082] 3. KRDHMVLHESVNAAGIT (SEQ ID NO: 11)

[0083] 4. RDHMVLHEYVNAAGIT (SEQ ID NO: 12)

[0084] 5. GDAVQIQEHAVAKYFTV (SEQ ID NO: 13)

[0085] 6. GDTVQLQEHAVAKYFTV (SEQ ID NO: 14)

[0086] 7. GETIQLQEHAVAKYFTE (SEQ ID NO: 15)

[0087] or a truncated version thereof. Truncation can include shortening by up to 5 amino acids from the N-terminus, C-terminus, or a combination thereof.

[0088] GFP 11 or GFP 1-10 may be fused to a protein of interest via an amino acid linker. In one embodiment, the oligopeptide linker, peptide linker, or polypeptide linker can be 0-50 amino acids.

[0089] Also disclosed are nucleic acid sequences for expressing a particular tag. The nucleic acid sequences include

[0090] SEQ ID NO: 16

[0091] 5’GGTGATACCGTTCAGCTGCAAGAACATGCAGTTGCAAAATACTTTACCGTG

[0092] SEQ ID NO: 17

[0093] 5’GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA

[0094] or a truncated version thereof.

[0095] These sequences can be repeated one or more times to produce a protein with multiple GFP 11 domains.

[0096] For example, sfCherry 1-10 The polypeptide amino acid sequence can be:

[0097] SEQ ID NO: 18

[0098] MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLLGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDITSHNED

[0099] The complementary sfCherry 11 peptide amino acid sequence can be:

[0100] SEQ ID NO: 19

[0101] YTIVEQYERAEGRHSTGG

[0102] sfCherry 11 or sfCherry 1-10 The NFAST polypeptide amino acid sequence can be fused to a protein of interest via an amino acid linker. In one embodiment, the oligopeptide linker, peptide linker, or polypeptide linker can be 0-50 amino acids.

[0103] For example, the NFAST polypeptide amino acid sequence can be:

[0104] SEQ IS NO: 20

[0105] MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKVKVHMKKALS

[0106] The complementary CFAST 11 The peptide amino acid sequence can be:

[0107] SEQ ID NO: 21

[0108] GDSYWVFVKRV

[0109] or the complementary CFAST 10 The peptide amino acid sequence can be:

[0110] SEQ ID NO: 22

[0111] GDSYWVFVKR

[0112] NFAST, CFAST 11 and / or CFAST 10 The NFAST polypeptide amino acid sequence can be fused to a protein of interest via an amino acid linker. In one embodiment, the oligopeptide linker, peptide linker, or polypeptide linker can be 0-50 amino acids.

[0113] If desired, the tag can be cleaved from the POI for tag / detection agent removal. Cleavage can be performed using, for example, a protease or a metal cation.

[0114] Expression can be performed using cell-free protein synthesis reagents derived from whole cell extracts. Expression can be performed using cell-free protein synthesis reagents derived from reconstituted systems comprising assembled components for transcription and translation in a system of purified recombinant elements (PURE).

[0115] Any of the methods described herein can be performed in, for example, a microtiter plate or a microcentrifuge tube. Any of the methods can be performed on a microfluidic or digital microfluidic device. The digital microfluidic device can comprise an oil-filled environment or a humidified gas environment, wherein the humidified gas environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.

[0116] A method of expressing proteins in droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes is disclosed, wherein the proteins have ccGFP 11 peptide amino acid sequence tags, wherein one portion of the droplets comprises ccGFP during the expression process 1-10 , another portion has ccGFP added after expression 1-10 , and comparing the levels of ccGFP 1-11 signal from the droplets.

[0117] Binding moieties for purification can comprise four or more amino acids. Binding sequences can comprise 4-30 amino acids. Binding moieties can be selected from the following:

[0118] Alpha tag (SRLEEELRRRLTE) (SEQ ID NO: 23)

[0119] Avi tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 24)

[0120] C tag (EPEA) (SEQ ID NO: 25)

[0121] Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 26)

[0122] Dog tag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 27)

[0123] E tag (GAPVPYPDPLEPR) (SEQ ID NO: 28)

[0124] FLAG (DYKDDDDK) (SEQ ID NO: 29)

[0125] G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 30)

[0126] HA (YPYDVPDYA) (SEQ ID NO: 31)

[0127] His (HHHHHH) (SEQ ID NO: 32)

[0128] Isopipeptide tag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33)

[0129] Lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)

[0130] Myc (EQKLISEEDL) (SEQ ID NO: 35)

[0131] NE tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 36)

[0132] Polyglutamate tag (EEEEEEE) (SEQ ID NO: 37)

[0133] Polyarginine tag (RRRRRRR) (SEQ ID NO: 38)

[0134] Rho1D4 tag (TETSQVAPA) (SEQ ID NO: 39)

[0135] SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 40)

[0136] Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 41)

[0137] SH3 (STVPVAPPRRRRG) (SEQ ID NO: 42)

[0138] SNAC (GSHHW) (SEQ ID NO: 43)

[0139] Snooptag (KLGDIEFIKVNK) (SEQ ID NO: 44)

[0140] Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 45)

[0141] Softag 3 (TQDPSRVG) (SEQ ID NO: 46)

[0142] Spot tag (PDRVRAVSHWSS) (SEQ ID NO: 47)

[0143] Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 48)

[0144] S tag (KETAAAKFERQHMDS) (SEQ ID NO: 49)

[0145] Strep tag (AWAHPQPGG) (SEQ ID NO: 50) (AWRHPQFGG) (SEQ ID NO: 51)

[0146] Strep tag II (WSHPQFEK) (SEQ ID NO: 52)

[0147] T7 tag (MASMTGGQQMG) (SEQ ID NO: 53)

[0148] TC tag (EVHTNQDPLD) (SEQ ID NO: 54)

[0149] Ty tag (CCPGCC) (SEQ ID NO: 55)

[0150] VSV tag (YTDIEMNRLGK) (SEQ ID NO: 56)

[0151] Xpress tag (DLYDDDDK) (SEQ ID NO: 57).

[0152] A method is disclosed comprising expressing one or more proteins in one or more droplets on a digital microfluidics device having a two-dimensional planar microelectrode array, wherein the proteins have ccGFP 11 peptide amino sequence tag and Strep tag, splitting the droplet into at least two volumes, after the expression process, merging the droplet containing ccGFP 1-10 into one of the droplets and measuring the uniformity of the fluorescence signal to determine the degree of aggregation of the expressed protein, taking the corresponding droplet containing the protein that has been expressed soluble but not ccGFP 1-10 and adding magnetic beads that bind to the strep tag, wherein the droplet with the magnetic beads has been merged and split one or more times such that each final split droplet contains a uniform concentration of suspended beads, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, after the elution process adding a droplet containing ccGFP1-10 droplets and measure the fluorescent signal to determine the level of protein purification and to determine the conditions under which purified soluble protein is obtained.

[0153] The method can be performed in parallel on different sequences. The method can use at least 8 different nucleic acid templates that can be screened against at least 4 different expression reagents on the same device. The device is capable of handling multiple droplets in parallel, for example, the device can manipulate at least 192 droplets individually.

[0154] The expression comparison and purification screen can identify the optimal conditions to express and purify the desired protein in the most soluble and most stable form.

[0155] BRIEF DESCRIPTION OF DRAWINGS

[0156] Figure 1 The target protein with the peptide tag (detection tag) is shown in the presence of the protein bound to the peptide tag (detection protein). In the case where the detection tag and detection protein produce a detectable signal upon binding, then the amount of target protein can be measured.

[0157] Figure 2 An animated illustration of protein expression showing different proteins with different levels of soluble yield and aggregation. Some conditions have no expression (dark color). Some conditions produce high levels of soluble protein (white squares of uniform intensity). Some conditions produce protein aggregates, meaning that the expressed protein is insoluble and clumped together (white dots in dark background). Some produce a mixture of soluble protein and aggregated protein (white dots in gray background). Thus, the level of soluble expression and the level of aggregation can be determined by adding a detection species to the expressed protein.

[0158] Figure 3 A schematic workflow showing protein expression. DNA linear expression constructs (LEC) can be mixed with expression reagents and combined. The mixed reactants can be split into multiple aliquots and subjected to protein expression. After expression has occurred, a solubility detection species (DET) is added to one of the droplets. When the expressed protein is soluble and has low levels of aggregation, the signal is uniform, indicating that the protein is expressed solubly.

[0159] Figure 4 A schematic workflow showing protein expression. DNA linear expression constructs (LEC) can be mixed with expression reagents and combined. The mixed reactants can be split into multiple aliquots and subjected to protein expression. After expression has occurred, a solubility detection species (DET) is added to one of the droplets. When the expressed protein is aggregated, the detection agent gives a non-uniform signal, indicating that the expressed protein is insoluble or aggregated to some extent. Insoluble or aggregated POI is less likely to be purified from the droplet.

[0160] Figure 5 Experimental results for 24 different proteins expressed in reconstituted cell-free protein synthesis systems in droplets displayed on an electrowetting on dielectric (EWoD) device. Each construct contained GFP 11 tag. In the row labeled "Screening," the GFP 1-10 detection agent was present from the start of expression. The row labeled "End-point" shows the fluorescence signal after 10 hours of expression in the absence of the GFP 1-10 detection agent, followed by 5 hours of complementation with the GFP 1-10 detection agent. This experiment shows a significant difference between expression / complementation in the "Screen BioInk" compared to the "End-point" detection. The detection of protein clusters formed after expression with only end-point detection means that the proteins are aggregating after expression, reducing the soluble yield. It is evident from the images that several constructs have spots, indicating that protein aggregates are likely present. The level of aggregation enables the identification of conditions worth further testing as well as the identification of proteins with high levels of aggregation for which further purification is unlikely to yield material.

[0161] Figure 6 An illustrative workflow showing protein expression and purification. DNA linear expression constructs (LECs) can be mixed with expression reagents and combined. The mixed reaction can be split into multiple aliquots and subjected to protein expression. After expression has occurred, a droplet is added with a soluble detection agent (DET). If the expressed protein aggregates, the detection agent produces a non-uniform signal, indicating that the expressed protein is to some extent insoluble or aggregated. Insoluble or aggregated POIs are less likely to be purified from the droplet, and such droplets are not taken for purification. If the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, indicating that the protein is expressed soluble. Such droplets or a subset of them are taken for purification. A droplet containing beads can be added to capture the expressed POI with a binding tag. The beads can be immobilized and washed by adding additional droplets and immobilizing the beads, while removing the droplets. The bound material can be eluted from the magnetic beads by breaking the binding. The process of adding detection agent can be repeated. When a strong signal is seen from the soluble material, the protein has been purified and eluted. If the signal is weak, missing, or aggregated, the soluble purified POI obtained is limited. The results of detection agent screens 1 and 2 can be compared to establish conditions for expression and purification.

[0162] Figure 7 An illustrative workflow showing protein expression and purification. DNA linear expression constructs (LECs) can be mixed with expression reagents and combined. The mixed reaction can be split into multiple aliquots and subjected to protein expression. After expression has occurred, a droplet is added with a soluble detection agent (DET). If the expressed protein aggregates, the detection agent produces a non-uniform signal, indicating that the expressed protein is to some extent insoluble or aggregated. Insoluble or aggregated POIs are less likely to be purified from the droplet, and such droplets are not taken for purification. If the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, indicating that the protein is expressed soluble. Such droplets or a subset of them are taken for purification. A droplet containing beads can be added to capture the expressed POI with a binding tag. The beads can be immobilized and washed by adding additional droplets and immobilizing the beads, while removing the droplets. The bound material can be eluted from the magnetic beads by breaking the binding. The process of adding detection agent can be repeated. When a strong signal is seen from the soluble material, the protein has been purified and eluted. If the signal is weak, missing, or aggregated, the soluble purified POI obtained is limited. The results of detection agent screens 1 and 2 can be compared to establish conditions for expression and purification. Figure 6Animation of workflow. Once optimal conditions are identified, scale-up can be used to produce protein to obtain larger volumes of material. For example, 24 DNA constructs can be screened under 8 different conditions to determine optimal expression conditions. The 24 constructs can be 24 independent sequences for different POIs or a single POI with 24 different flanking (solubility tags, etc.). The 8 conditions can be lysates or reconstituted systems with different components. Once the optimal expression system is identified, reagents can be selected for purification screening. For example, screening can select 30 liquid volumes (droplets) to which beads are added. Scale-up can be performed in tubes outside of the device to provide conditions for optimal purification to achieve soluble protein.

[0163] Figure 8 Expression screen of VEGF proteins with different flanking sequences. An expression and solubility screen was first performed to determine optimal expression of human VEGF using three different cell-free reagents in combination with constructs containing 8 different nucleic acid template variants corresponding to VEGF with different solubility tags at the N- and C-termini. The three cell-free mixtures were composed of different cell-free protein synthesis reagents. Mixture 3 was designed to promote disulfide bond formation, while mixture 4 contained molecular chaperones to promote protein folding. Mixture 2 contained the core components of the cell-free synthesis reagents without any additional components. Each construct was run twice. A total of 192 droplets were run on a single cartridge. Dotted lines indicate the cell-free mixture run with the corresponding construct. The location of the detector (DET) is also noted. Bright white fluorescent spots indicate expression of soluble protein.

[0164] Figure 9 Graphical representation of spot intensity in Figure 8 Concentration of expressed protein was calculated based on measured fluorescence intensity. Construct A5 (SUMO_VEGF_STREP_DET) produced the highest expression in mixture 3. The construct with the highest yield (mg / mL) was selected for on-cartridge purification (highlighted by green dotted box).

[0165] Figure 10 Image showing 30 soluble candidates that exhibited the highest expression yield were automatically selected and continued to the on-cartridge purification step. These candidates included SUMO-VEGF expressed in mixture 2, mixture 3, and mixture 4. These expression candidates were purified on-cartridge using magnetic beads. Figure 5 Location of SUMO_VEGF_STREP_DET purified on-cartridge is shown. Fluorescence intensity measurements were used to determine the yield of purified protein.

[0166] Figure 11 Graphical representation of spot intensity in Figure 10Graphical representation of the SUMO-VEGF-STREP_DET expression and purification yield is shown and a side-by-side comparison of the expression and purification yield is shown. The results show that the construct was soluble and purifiable in all three cell-free mixtures. Mixture 3 produced the highest protein expression and purification yield and was therefore selected for scale-up expression of the out-of-cassette.

[0167] Figure 12 Gel showing out-of-cassette, scale-up expression (500 μΐ cell-free protein synthesis reaction) and purification of SUMO_VEGF_STREP_DET. The SDS-PAGE shows that the VEGF protein has a slightly higher molecular weight than the predicted molecular weight of 36.32 kDa, as the SUMO tag typically exhibits a higher molecular weight than its calculated molecular weight on SDS-PAGE gels. Lane 1 molecular weight standard reference, lane 2 mixture 3 negative control, lane 3 total CFPS after expression, lane 4 CPFS obtained after centrifugation (soluble) and soluble fraction obtained, lane 5 unbound; flow-through sample not bound to beads, lane 6 purified (elution); sample eluted from resin beads.

[0168] Figure 13 shows an 8x4 droplet array, where each cycle the droplets are repositioned. The magnetic particles are visualized and quantified by fluorescence imaging of the fluorescein bound to the surface of the magnetic particles. Two devices are shown, which have a 32x array of droplets containing magnetic beads microparticles distributed in 49 px 2 droplets in an 8x4 pattern.

[0169] Figure 14 shows the rearrangement of the droplets to get a homogeneous mixture. Each array goes through the indicated flow for several cycles (40 cycles in this case), where the droplets containing magnetic beads are continuously mixed and split.

[0170] Figure 15 shows the fluorescence images of the two arrays after the cycles.

[0171] Figure 16 shows a schematic of the mixing process of the top 8 droplets.

[0172] Figure 17 shows the homogenization and the distribution of the size of the bead clusters. The bead cluster size represents the number of beads in each droplet, as the beads are immobilized to the surface. DETAILED DESCRIPTION

[0174] Applicants have realized that purification of protein sequences is unpredictable and requires screening. Just because a protein is expressed does not mean that it can be obtained in a soluble purified form. The level of aggregation of a protein after expression is an important measure of whether the protein can be subsequently purified. High levels of protein aggregation can indicate a lack of soluble protein for purification. Disclosed herein is a method for determining the degree of aggregation of a protein expressed in a cell-free system, comprising expressing a protein of interest (POI) in a droplet on a digital microfluidics device, wherein the expressed POI is measured by an optically detectable signal, and the level of insoluble POI is measured.

[0175] To accurately measure and compare the amount of purified protein, a large number of droplet volumes with the same number of beads is required. The lack of uniformity in bead distribution between droplets introduces measurement error in the amount of purified protein that is independent of the starting protein concentration. The assay developed by the inventors requires uniform beads in multiple droplets.

[0176] The non-uniform distribution can be normalized by repeated mixing and splitting. For example, electrowetting control allows two or more droplets to be merged and split. If all droplets are merged and split with other droplets, any differences between the initial droplet concentrations are reduced or eliminated. Such uniform droplets can be used in the assay to measure the success rate of purification without worrying about the lack of starting uniformity of the beads used for purification.

[0177] A method is disclosed that relies on parallel processing of fluid volumes. Described herein is a method of producing aqueous droplets with uniformly concentrated suspended beads, wherein the method comprises:

[0178] a. a reservoir containing an aqueous liquid with beads suspended therein,

[0179] b. forming a plurality of droplet subvolumes from the reservoir,

[0180] c. merging two or more droplets,

[0181] d. splitting the merged droplet, and

[0182] e. repeating steps c and d one or more times such that each final split droplet contains a uniformly concentrated suspension of beads.

[0183] Described herein is a method of producing aqueous droplets with uniformly concentrated suspended beads, wherein the method comprises:

[0184] a. a reservoir containing an aqueous liquid with beads suspended therein,

[0185] b. forming at least four droplet subvolumes from the reservoir,

[0186] c. merging each droplet with at least one other droplet,

[0187] d. splitting the merged droplet to form at least four additional droplets, and

[0188] e. repeating steps c and d one or more times such that each final split droplet contains a uniform concentration of suspended beads.

[0189] A method for synthesizing, characterizing, and purifying one or more proteins having a detection tag and a binding tag is disclosed, the method comprising the steps of:

[0190] i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes;

[0191] iia splitting the merged volumes into at least two aliquots and effecting protein expression in at least two aliquots; or

[0192] iib effecting protein expression and then splitting into at least two aliquots;

[0193] iii adding to at least one aliquot a detection reagent that binds to the detection tag to determine protein expression levels and conditions under which the protein is expressed in soluble form;

[0194] iv selecting a volume in which the protein has been expressed in soluble form and adding to a subpopulation of aliquots magnetic beads, wherein the beads bind to the binding tag and the beads in the droplets have been merged and split one or more times such that each final split droplet contains a uniform concentration of suspended beads;

[0195] v separating the magnetic beads from the expression reagents;

[0196] vi optionally adding to the magnetic beads a wash reagent to remove unbound material;

[0197] vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and

[0198] viii adding to at least one eluted aliquot a detection reagent that binds to the detection tag to determine protein purification levels and conditions under which purified soluble protein is obtained.

[0199] When expressing proteins, various parameters are important in determining successful expression, both in terms of yield and function. One of the factors is the soluble yield of the expressed protein, as many proteins are expressed in insoluble form, or become inactive due to lack of stability.

[0200] The determination of soluble yield described herein includes expression and detection of the expressed protein to measure the degree of aggregation. The protein of interest (POI) can have a detection tag that binds to a detection agent to produce a signal. The detection agent and / or the POI can have additional solubility factors.

[0201] The method can be used as part of a process to determine if the expressed protein is suitable for subsequent purification testing. For example, the method can include expressing a protein of interest (POI) in a first reagent volume and splitting the reagent volume into multiple aliquots. The splitting can be performed before or after the expression process. The protein of interest can have a binding tag that complements a detection substance and becomes fluorescent. The detection substance is added to one or more of the aliquots and the signal is recorded. The uniformity of the signal in the droplet determines the degree of aggregation. When the signal is uniform within the droplet, the POI is soluble with little or no aggregation. When the signal is non-uniform in the droplet, seen in clumps, the protein is aggregated and insoluble. Thus, the level of the overall signal indicates the level of expression, while the signal uniformity determines the aggregation. Thus, the level of expression and the level of soluble expression can be determined by viewing the amount and uniformity of the signal within the droplet. The degree of aggregation can be measured by using a uniformity measure, the number of aggregates, the area of aggregates, the intensity count of aggregates, or by using a dispersion measure.

[0202] If the protein is unstable or difficult to solubilize, the protein expression can produce aggregation. Protein aggregates result in a lack of uniform signal for the detection agent. Thus, a POI with a high soluble yield produces a uniform level of signal, a protein with a low soluble yield can have a low or high level of signal, but the signal is aggregated and non-uniform throughout the reaction volume due to the aggregation of the protein.

[0203] A determination of protein stability and further potential for purification can be performed on the device. Further protein stability determinations can include

[0204] • solubility

[0205] • aggregation (over time)

[0206] • thermal stability.

[0207] Protein thermal stability does not necessarily predict the rate of aggregation at low temperatures (and vice versa). For example, the melting temperature (Tm) of a monoclonal antibody does not necessarily correlate with the rate of aggregation of the monoclonal antibody at room temperature (Mol. Pharmaceutics 2016, 13, 307−319). At high temperatures, the rate of aggregation depends on the unfolded state, while at low temperatures, the rate of aggregation depends on the native state. Thus, denaturing a protein can or can not give an accurate measure of the stability of the protein in its native state. m The device can be used to determine the stability of a protein at low temperatures. The device can be used to determine the stability of a protein at room temperature. The device can be used to determine the stability of a protein at high temperatures.

[0208] It would be very useful to provide a measure of protein aggregation over time, providing additional information beyond simply providing a measure of expression yield. Producing stable protein formulations is a key to protein development and manufacturing. Since proteins can be exposed to various types of stress, such as temperature, pH, salt, mechanical stress, surface interactions, or oxidation, it is critical that the formulation be robust against external stress factors. Protein aggregation is a common problem faced during expression. Therefore, it is highly desirable to predict or control protein aggregation during production of a protein of interest. Described herein is a high-throughput assay for protein aggregation / stability that uses directly expressed material to measure protein stability.

[0209] Protein purification can be challenging and time consuming, often resulting in substantial loss of material or failure to obtain the material in purified form. Therefore, it is desirable to measure protein stability directly after expression without having to purify the protein from the protein used in the expression. Using optical-based analysis techniques, it is possible to measure in crude cell lysates or directly in cell-free expression systems, as these techniques can tolerate the presence of other proteins that would otherwise be stable or otherwise undetectable to the conditions being measured.

[0210] Disclosed is a method for protein synthesis comprising expressing a protein, wherein the expressed protein contains a subcomponent of a fluorescent protein, the method comprising monitoring the stability of the protein over time by producing the fluorescent protein at different times in different batches of material, such as before and after purification.

[0211] The method for measuring stability avoids the need for complex purification steps or gel-based separations prior to performing the assay. The fluorescence-based assay can be used to measure the amount of expressed protein (soluble yield) in conjunction with measuring protein stability. Therefore, both yield and stability can be determined after expression without the need to run a gel or purify the material.

[0212] The fluorescence measurement can be used as a real-time measure of soluble protein expression. When a tag sequence is produced, the presence of the remaining fluorescent protein as a detection species enables real-time measurement of the amount of tag sequence and soluble protein. Alternatively, fluorescence can be measured by adding a detection species after expression to measure insoluble protein. During the stability assay, fluorescence can be retained and monitored. Alternatively, additional detection protein can be added after the stability step to determine the level of protein remaining. Therefore, both the yield of synthesis and the stability of the expressed protein can be determined. For example, GFP 11 may be attached to the expressed protein, GFP 1-10 is used as the detection species.

[0213] This screening workflow enables users to rapidly screen different expression systems in the form of cell-free lysates or reconstituted systems. After the best expression system is identified, the soluble yield, stability, and purification conditions can be directly measured on the same device.

[0214] The protein can be expressed with a binding tag. The binding moiety can be a region of an amino acid / peptide sequence. The affinity binding site can be a region of an amino acid / peptide sequence specific for a particular antibody. The tag can be attached to the N- or C-terminus. For example, the binding moiety can be selected from the following exemplary list of peptide affinity binding sites:

[0215] Alpha tag (SRLEEELRRRLTE) (SEQ ID NO: 23)

[0216] Avi tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 24)

[0217] C tag (EPEA) (SEQ ID NO: 25)

[0218] Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 26)

[0219] Dog tag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 27)

[0220] E tag (GAPVPYPDPLEPR) (SEQ ID NO: 28)

[0221] FLAG (DYKDDDDK) (SEQ ID NO: 29)

[0222] G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 30)

[0223] HA (YPYDVPDYA) (SEQ ID NO: 31)

[0224] His (HHHHHH) (SEQ ID NO: 32)

[0225] Isopipe tag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33)

[0226] Lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)

[0227] Myc (EQKLISEEDL) (SEQ ID NO: 35)

[0228] NE tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 36)

[0229] Polyglutamate tag (EEEEEEE) (SEQ ID NO: 37)

[0230] Polyarginine tag (RRRRRRR) (SEQ ID NO: 38)

[0231] Rho1D4 tag (TETSQVAPA) (SEQ ID NO: 39)

[0232] SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 40)

[0233] Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 41)

[0234] SH3 (STVPVAPPRRRRG) (SEQ ID NO: 42)

[0235] SNAC (GSHHW) (SEQ ID NO: 43)

[0236] Snooptag (KLGDIEFIKVNK) (SEQ ID NO: 44)

[0237] Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 45)

[0238] Softag 3 (TQDPSRVG) (SEQ ID NO: 46)

[0239] Spot tag (PDRVRAVSHWSS) (SEQ ID NO: 47)

[0240] Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 48)

[0241] S tag (KETAAAKFERQHMDS) (SEQ ID NO: 49)

[0242] Strep tag (AWAHPQPGG) (SEQ ID NO: 50) (AWRHPQFGG) (SEQ ID NO: 51)

[0243] Strep tag II (WSHPQFEK) (SEQ ID NO: 52)

[0244] T7 tag (MASMTGGQQMG) (SEQ ID NO: 53)

[0245] TC tag (EVHTNQDPLD) (SEQ ID NO: 54)

[0246] Ty tag (CCPGCC) (SEQ ID NO: 55)

[0247] VSV tag (YTDIEMNRLGK) (SEQ ID NO: 56)

[0248] Xpress tag (DLYDDDDK) (SEQ ID NO: 57)

[0249] The binding moiety can comprise a small molecule affinity tag, such as biotin. The binding moiety can comprise a specific nucleic acid sequence.

[0250] The expressed amino acid sequence can be bound to a bead with affinity for the tag. Suitable beads can be magnetic or paramagnetic beads. The beads can have, for example, metal ions chelated to polyhistidine, or streptavidin or modified streptavidin bound to a strep tag. The beads can be of a size that is manipulable within a droplet, for example an average diameter of 1-50 μιη. The beads can have a magnetic core and a polymeric coating. The beads can be, for example, silica or agarose. Suitable magnetic beads are commercially available.

[0251] The beads can be incorporated into the droplet on the device. When exposed to a magnetic field, the beads can be immobilized while the droplet can be moved or split. Thus, the beads can be removed from unbound reagents. A washing step can be performed by adding further reagents, agitating the beads in the droplet, immobilizing the beads and removing the droplet.

[0252] The binding can be reversed using a suitable buffer, for example a buffer containing biotin to release a strep tag or imidazole to release a polyhistidine.

[0253] The process can be performed in droplets, which can be manipulated by electrokinetics for protein expression and analysis. Any electrokinetic means can be used to move the droplets. Electro wetting on dielectrics (EWoD) can be used to move the droplets. The electrical signal on an EWoD or optical EWoD device can be delivered by segmented electrodes, active matrix thin film transistors or digital micromirrors.

[0254] Cell-free expression of peptides or proteins can be achieved using cell lysates or reconstitution systems equipped with reagents for protein expression. Common components of cell-free reactions include an energy source, an amino acid supply source, cofactors such as magnesium, and associated enzymes. Cell extracts are obtained by lysing target cells and removing cell walls, DNA, genomes, and other debris by centrifugation. The remainder consists of cellular machinery, including ribosomes, aminoacyl-tRNA synthetase, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the template can be expressed as a peptide or protein using cell-derived expression machinery.

[0255] To optimize expression, the expression system can be supplemented with additional components, including purified enzymes. These additional components may include salts, cofactors, buffers, surfactants, molecular chaperones, or other protein components. The additional protein components may be selected from, for example, molecular chaperones, glycosylation enzymes, proteases, redox-active enzymes, phosphorylases, and kinases.

[0256] Expression compositions can be assembled on the device by mixing various droplets to allow for parallel screening of various compositions.

[0257] As an example, screening reagents may include:

[0258] ■ Molecular chaperone mixtures (e.g.) PUREfrex GroE mix )

[0259] ■ Kinase 1 (e.g.) NEB CK2 )

[0260] ■ Kinase 2 (e.g.) NEB PKA )

[0261] ■ Protease 1 (e.g.) NEB TEV )

[0262] ■ Protease 2 (e.g.) Merck HRV 3C )

[0263] ■ Common metal ion mixtures

[0264] ■ Common auxiliary factor mixtures.

[0265] Users can blend the compositions and monitor the expression level of the target protein under each blending condition.

[0266] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used for cell-free protein synthesis (CFPS) include plasmids, linear expression constructs (LECs), and mRNA. Plasmids are circular templates that can be produced in cells or by synthesis. LECs can be prepared via PCR. mRNA can be produced by in vitro transcription systems. The method can use a single nucleic acid template per droplet. The method can use multiple nucleic acid templates per droplet. The method can use multiple droplets each having a different nucleic acid template.

[0267] Energy sources are an important part of cell-free reactions. Typically, a separate mixture containing the desired energy source, along with a 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 replenished by further adding reagents to the droplets during the process.

[0268] A cell-free extract with components for protein expression contains everything needed for protein expression except for the nucleic acid template. Thus, the term includes all relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions, and energy sources. Once the nucleic acid template is added, expression of the protein begins, and no other reagents are needed.

[0269] Thus, before the template is added, the cell lysate can be supplemented with additional reagents. A cell-free extract with components for protein expression will typically be produced as a bulk reagent or'master mix' that can be dispensed into many identical droplets before different templates are added to different droplets, respectively. Common cell extracts used currently are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE), and Kluyveromyces yeast (D2P system). All of these extracts are commercially available.

[0270] A cell-free system can be assembled from the required reagents without coming from a cell extract. Systems based on reconstituted, purified molecular reagents are commercially available, such as the PURE system for producing proteins, which can be used as provided. The PURE system consists of all enzymes involved in transcription and translation, as well as highly purified 70S ribosomes. Protein synthesis reactions of the PURE system lack proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

[0271] Rapid screening of various variable factors is achieved using a population of droplets with different components to identify the optimal conditions for expressing a desired protein. The protein can comprise in a sequence with other amino acid domains, such as a soluble factor or a binding tag.

[0272] Flanking sequences attached to the target sequence may contain elements required for transcription and translation, such as promoter regions including the T7 promoter binding site, ribosome binding site, start codon, and stop codon. Flanking sequences may also optionally contain elements such as solubility tags, purification tags, or detection tags.

[0273] The protein-soluble sequence can be linked to the detection reagent and the POI sequence. For example, GFP... 1-10 It can be linked to other elements to improve solubility. The solubility-enhancing sequence can be a peptide sequence or a naturally occurring sequence. The solubility-enhancing sequence can be selected from, for example, maltose-binding protein (MBP), small ubiquitin-like modified protein (SUMO), glutathione S-transferase (GST), or thioredoxin (TRX). The tag can be linked to the C-terminus or N-terminus. Examples of any solubility enhancer can be used. A list of possible proteins is shown below. Any sequence selected from the following list can be chosen:

[0274]

[0275]

[0276] Any fluorescent protein can be used. The term GFP is used in this document to describe a group of green fluorescent proteins from different organisms, including proteins sfGFP, GFP, eGFP, ccGFP, deGFP, and frGFP. Fluorescent proteins can be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, and miRFP670nano. For example, the peptide tag can be GFP. 11 The other polypeptide is GFP. 1-10 The peptide tag can be a component of sfCherry. 11 The other peptide is sfCherry 1-10 In the presence of hydroxybenzyl methylene rhodanine analogues, peptide tags can be CFAST. 11 or CFAST 10 The other polypeptide is CFAST. The peptide tag can be ccGFP. 11 The other polypeptide is ccGFP. 1-10 .

[0277] The fluorescent protein can be GFP. The fluorescent protein can be sfGFP. The fluorescent protein can be ccGFP. The solubility-enhancing component can contain ccGFP. 1-10and MBP.

[0278] As the expressed protein binds to the binding partner, the protein can be assembled and thereby become fluorescent. The affinity interaction causes the two subcomponents of the fluorescent protein to be close enough to one another to bind and induce fluorescence.

[0279] complementary GFP 11 The peptide amino acid sequence tag can be as follows:

[0280] 1. KRDHMVLLEFVTAAGITGT

[0281] 2. KRDHMVLHEFVTAAGITGT

[0282] 3. KRDHMVLHESVNAAGIT

[0283] 4. RDHMVLHEYVNAAGIT

[0284] 5. GDAVQIQEHAVAKYFTV

[0285] 6. GDTVQLQEHAVAKYFTV

[0286] 7. GETIQLQEHAVAKYFTE.

[0287] or a truncated sequence thereof, or a truncated version thereof. Truncation can include shortening by up to 5 amino acids from the N-terminus, the C-terminus, or a combination thereof.

[0288] The properties of the expressed protein can be characterized on the device. Initial screening can be based on the level of soluble expression, by measuring fluorescence formed when a detection agent is complementary to the expressed sequence. The protein can remain fluorescent during immobilization, at which point the level of affinity purification can be determined. Alternatively, the non-fluorescent portion of the protein can be purified, and a detection agent added to measure the soluble yield of the purified material.

[0289] A method is disclosed of taking a plurality of droplets of a protein having a GFP 11 tag, and monitoring changes in the level of GFP 11 tag to determine the yield of protein expression.

[0290] The method can measure aggregation by measuring solubility before and after purification. For example,

[0291] 1. A parent droplet containing a nucleic acid template and protein expression reagents expresses a POI.

[0292] 2. (Before, during, or after expression) the parent droplet is split into a plurality of child droplets.

[0293] 3. Add droplet containing detection agent to sub-droplet 1.

[0294] 4. Select subset of sub-droplets 2, add magnetic beads, bind expressed protein.

[0295] 5. Wash beads to remove unbound material.

[0296] 6. Remove POI from beads.

[0297] 7. Add another droplet containing detection agent to eluted POI.

[0298] 8. Complement (7) and (3), each of the same volume and time period.

[0299] 9. Measure fluorescence of both droplets and compare. Measure protein solubility yield before and after purification to determine purification yield.

[0300] Once the protein is expressed, expression can be stopped by the addition of a chemical agent to prevent further expression. The assay for purification should be independent of further expression, as increasing the amount of protein by additional protein expression can bias the measurement of purification. For example, the detection solution can be mixed with an agent that prevents protein synthesis, such as Mg++ chelator (EDTA), to achieve integration of stopping and detection. Alternatively, the chelator / EDTA can be introduced into all sub-droplets first, and the detection agent introduced at a different time point.

[0301] Screening and analysis can be performed in liquid reagent volumes, for example in microtiter plates or strip tubes. The reagent volumes can be split so that part is tested and part is reserved for later use.

[0302] Such screening, characterization, and purification can all be performed on a single device, which can be a digital microfluidic device. The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term digital microfluidic device excludes any device that merely has droplets in a flow of oil in a channel. By activating specific electrodes, droplets move over the surface by electrokinetic forces. Upon electrode activation, the wettability of the hydrophobic dielectric increases, thus causing the droplet to spread over the surface. The setup of a digital microfluidic (DMF) device 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.

[0303] 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 a large amount of oil.

[0304] Digital microfluidics (DMF) refers to a two-dimensional planar platform for lab-on-a-chip systems that is based on 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 with analytical analysis processes such as mass spectrometry, colorimetry, electrochemistry, and electrochemiluminescence.

[0305] Any means of electrokinetic movement can be used to move the droplets. Aqueous droplets can be moved using electrowetting on dielectrics (EWoD). Electrowetting on dielectrics (EWoD) is a variant of the electrowetting phenomenon based on dielectric materials. In the EWoD process, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon electrode activation, the wettability of the aqueous droplet on the hydrophobic dielectric increases, thus causing the droplet to spread out on the surface.

[0306] The electrical signal on EWoD or optically activated amorphous silicon (a-Si) EWoD devices can be delivered by segmented electrodes, active matrix thin film transistors, or digital micromirrors. Optically activated s-Si EWoD devices are well known in the art for driving droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0307] The droplets can be supplied with a source of supplemental oxygen. For example, during protein expression, a droplet or bubble containing gaseous or dissolved oxygen can be merged with the aqueous droplet. Alternatively, the source of oxygen can be a molecular source that releases oxygen. Alternatively, the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen from the gaseous environment. Alternatively, the oil can be oxygenated.

[0308] The droplets can be formed prior to entering the microfluidic device and flow into the device. Alternatively, the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free system having components for protein expression to form a droplet.

[0309] Droplets can be driven on hydrophobic surfaces on 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, in particular 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). Superhydrophobic surfaces prevent biofouling compared to general fluorocarbon-based hydrophobic surfaces. Thus, superhydrophobic surfaces extend the ability of digital microfluidic devices to move CFPS droplets and universal 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 a porous PTFE membrane with Krtox-103 oil (DuPont) (Lab Chip, 2019, 19, 2275).

[0310] For electro-wetting on dielectric (EWoD), the change in reagent contact angle upon application of an electric potential is the inverse function of surface tension. Thus, for low voltage EWoD operation, the reduction in surface tension is achieved by the addition of surfactant to the reagent, which for CFPS reactions means addition to the lysate and addition to the DNA. This results in dilution of the lysate, which has been seen in experiments to result in a decrease in expression levels of the target protein. Thus, CFPS on DMF where surfactant is added to the solution being moved will necessarily result in dilution of the lysate and thus a decrease in protein expression levels. This further complicates the extrapolation of results on DMF to protein yield predictions in tubes, on top of being a problem in itself. A further disadvantage of having to add surfactant to the sample is that this 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. A further disadvantage of having to add surfactant to the sample is that certain downstream manipulations are hampered. For example, if the target protein is expressed in a cell-free system with a GFP 11 (or similar) peptide tag, this is hampered in the presence of surfactant. 1-10 Downstream manipulation to detect the polypeptide is hampered in the presence of surfactant.

[0311] Instead of adding high levels of surfactant to the aqueous sample, surfactant can be added to the oil, such as Span 85 (sorbitan trioleate). This has the advantage of allowing the CFPS reaction to be run on DMF without dilution or contamination. In addition, it simplifies the sample preparation process for setting up the reaction, improving ease of use and consistency of results. The use of 1% w / w Span 85 in dodecane allows for a non-diluted CFPS reaction on DMF and non-diluted detection of expressed non-fluorescent proteins. Other surfactants than Span 85 and oils than dodecane can be used. A range of concentrations of Span 85 can be used. The surfactant can be non-ionic, anionic, cationic, amphoteric. The oil can be a mineral or synthetic oil, including silicon oil, petroleum oil and perfluorinated oil. The surfactant can have a detrimental effect on (1) the efficiency of the CFPS reaction and (2) the detection system (if the detection system involves complementation of a tag and a detector). For example, by running the CFPS reaction on DMF with an oil-surfactant mixture, detection of the expressed protein can also be performed without dilution and without the addition of an aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the split-GFP system, so it can be beneficial to remove the surfactant from the reagent mixture and instead add it to the oil.

[0312] The fill liquid can be a hydrophobic or non-ionic liquid. For example, the fill liquid can be decane or dodecane. The fill liquid can be a silicon oil, such as dodecamethylpentasiloxane (DMPS). The fill liquid can contain a surfactant, for example a sorbitan ester, such as Span 85.

[0313] A method is disclosed which comprises expressing a plurality of droplets of a protein having a GFP 11 tag, and monitoring changes in the level of the GFP 11 tag for determining protein stability. A method is disclosed which comprises expressing a protein in droplets on a digital microfluidics device having a two-dimensional array of planar microelectrodes, wherein the protein has a GFP 11 peptide amino acid sequence tag, holding the droplets at a fixed temperature, and monitoring the droplets for changes in the level of the GFP 11 peptide amino acid sequence tag in the droplets.

[0314] A method is disclosed which comprises: expressing one or more proteins in one or more droplets on a digital microfluidics device having a two-dimensional array of planar microelectrodes, wherein the proteins have a ccGFP 11 peptide amino acid sequence tag and a Strep tag, splitting the droplets into at least two volumes, and detecting the ccGFP 1-10droplets containing soluble expressed protein but not ccGFP 1-10 droplets containing ccGFP and magnetic beads conjugated to strep-tag, beads are immobilized and washed to remove unbound material; POI is eluted by disrupting strep binding, after elution process droplets containing ccGFP 1-10 are added and fluorescence signal is measured to determine protein purification level and conditions to obtain purified soluble protein.

[0315] apparatus

[0316] Manipulation of droplets by application of electric potential can be achieved on electrodes covered with an insulator or dielectric or a series of insulators or dielectrics. Droplet manipulation resulting from the applied electric potential is known as electrowetting. Electrowetting occurs due to either a non-uniform electric field affecting the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on the solid surface (electrowetting on dielectrics or EWoD). DEP can also be used to generate forces on polarizable particles to induce their movement. The electrical signal can be transmitted to discrete electrodes, transistors, transistor arrays or semiconductor membranes, whose electrical properties can be modulated by optical signals.

[0317] EWoD phenomenon occurs when a droplet is 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 due to a change in the droplet contact angle. The electrowetting effect can be quantitatively treated with the Young-Lippmann equation:

[0318]

[0319] where θ0is the contact angle at zero electric field across the interface layer, γLGis the liquid-gas tension, c is the specific capacitance (given as ε r / t, where ε r is the dielectric constant of the insulator / dielectric, ε0is the vacuum permittivity and t is the thickness), V is the applied voltage or potential. Thus, the change in contact angle (inducing droplet movement) is a function of surface tension, potential, dielectric thickness and dielectric constant.

[0320] When a droplet is actuated by EWoD, two opposing sets of forces act on it: the electrowetting force induced by the electric field and the drag force, which includes the drag force and the contact line friction resulting from the droplet interaction with the filler medium (ref). The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is a function of the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε r 1 / 2 ​are variably determined. Thus, to reduce the driving 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 driving, 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 prone to damage before reaching the desired electro wetting contact angle large enough to drive the droplets. Thus, most academic research reports use much higher voltages >100V on thick dielectric films (>3 pm) that are easy to fabricate to achieve electro wetting.

[0321] However, high voltage EWoD based devices with thick dielectric films have limited industrial applicability, mainly because of their limited droplet multiplexing capability. 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 driving voltage of TFTs or optically activated a-Si is low (typically <15V). The bottleneck in manufacturing and thus adoption of low voltage devices has been the technical challenge of depositing high quality thin film insulators / dielectrics. Thus, there has been a particular need to improve the manufacturing and composition of thin film insulator / dielectric devices.

[0322] Generally, the electrodes (or array elements) used for EWoD are covered by (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 this material as a hydrophobic coating on the dielectric is generally < 100 nm and can have defects in the form of pinholes or porous structures; thus, it is particularly important that the insulator / dielectric be free of pinholes to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but due to its low dielectric constant and the thickness required to make it pinhole free, it has a high voltage requirement. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (such as SU-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Teflon is still used as a hydrophobic overcoat on these insulator / dielectric polymers due to minimal contact angle hysteresis and a large contact angle with aqueous solutions. However, there are difficulties in reliably fabricating pinhole-free coatings of < 1 micron of parylene or SU-8; thus, the thickness of these materials is generally kept at 2-5 microns at the expense of an increased voltage requirement for electrowetting. It has also been reported that traditional EWoD devices with parylene C are prone to breakage and instability for repeated droplet manipulation with cell culture media. Multilayer insulator devices with metal oxide and parylene C film deposition have been used to fabricate more robust insulator / dielectrics and enable operation at lower applied voltages. Inorganic materials such as metal oxides and semiconductor oxides, commonly used as "gate dielectrics" in the CMOS industry, have been used as insulator / dielectrics for EWoD devices. They offer the advantage of thin film deposition (< 100 nm) using standard cleanroom methods. These materials are inherently hydrophilic, require an additional hydrophobic coating, and are prone to pinhole formation due to the thin film layer deposition process. In conjunction with the need for lower voltage operation of EWoD, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using high dielectric constant insulator / dielectrics), (2) optimizing fabrication methods to make the insulator / dielectric pinhole free to avoid dielectric breakdown.

[0323] Operation of EWoD devices is affected by contact angle saturation and hysteresis, believed to be caused by any one or combination of the following phenomena: (1) charge trapping in the hydrophobic film or insulator / dielectric interface, (2) ion adsorption, (3) thermodynamic contact angle instability, (4) dielectric breakdown of the dielectric layer, (5) electrode-electrode-insulator interface capacitance (caused by double layer effects), and (6) fouling of the surface (such as by biological macromolecules to the surface). One of the detrimental effects of such hysteresis is a reduction in the operational lifetime of EWoD-based devices.

[0324] Contact angle hysteresis is believed to be a 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 at the insulator / dielectric can bring the liquid into contact with the electrode, leading to electrolysis. Hydrophobic insulators that are prone to pinholes or porosity further promote electrolysis.

[0325] Most studies on contact angle hysteresis on EWoD are based on short time scales and with low conductivity solutions. Long duration actuation (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCl) can 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 lead to (1) changes in the dielectric constant due to charge trapping (this is different from interface charging) and (2) changes in the surface potential of the pH-sensitive metal oxide. Both can lead to a decrease in the electrowetting forces that manipulate aqueous droplets, resulting in contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces the electrowetting on the electrode by suppressing the modulation of the contact angle when an electric field is applied, or makes it impossible to electrowet on the electrode.

[0326] An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each matrix electrode is coupled with 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.

[0327] The dielectric layer can 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 zirconium titanate, strontium titanate, or barium strontium titanate. The dielectric layer can have a thickness of 10 nm to 100 μιη. Combinations of more than one material can be used, and the dielectric layer can include more than one sublayer, which can be made of different materials.

[0328] The conformal layer can comprise parylene, siloxane, or an epoxy resin. It can be a thin protective parylene coating between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In the present invention, the reason for depositing parylene is not to improve insulating / dielectric properties, such as reducing pinholes, but to serve as a conformal layer between the dielectric layer and the hydrophobic layer. The inventors have found that parylene, as opposed to other similar insulating coatings of the same thickness, such as PDMS (polydimethylsiloxane), prevents contact angle hysteresis caused by high conductivity solutions or solutions that deviate from neutral pH for long periods of time. The thickness of the conformal layer can be 10 nm to 100 pm. The thickness of the conformal layer can be 100 nm to 200 nm.

[0329] The hydrophobic layer can comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite, a zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structures, a silica nanocoating, or a smooth liquid perfused porous coating.

[0330] The elements can include one or more of a plurality of array elements, each element containing an element circuit; a discrete electrode; a thin film semiconductor whose electrical properties are modulated by incident light; and a thin film photoconductor whose properties are modulated by incident light.

[0331] The functional coating can 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.

[0332] The electrokinetic device can include a controller to regulate the voltage provided to the individual matrix electrodes. The electrokinetic device can 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 operatively connected to the controller. This enables all individual elements to be controlled individually.

[0333] The second substrate can further comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates can be disposed such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electrokinetic working space between the hydrophobic layers.

[0334] The method is particularly suitable for aqueous droplets having a volume of 1 pL or less.

[0335] The EWoD-based devices shown and described below are active matrix thin film transistor devices containing thin film dielectric coatings with Teflon hydrophobic surface coatings. These devices are based on the devices described in the patent entitled “Digital microfluidic device comprising dual substrates with thin film transistors and capacitive sensing” filed by EInk Corporation, U.S. Patent Application No. 2019 / 0111433, which is incorporated by reference herein.

[0336] An electrokinetic device is described herein, comprising:

[0337] a first substrate having a matrix of electrodes, wherein each matrix electrode is coupled with a thin film transistor, and wherein the matrix electrodes are externally coated with a functional coating comprising:

[0338] a dielectric layer in contact with the matrix electrodes,

[0339] a conformal layer in contact with the dielectric layer, and

[0340] a hydrophobic layer in contact with the conformal layer;

[0341] a second substrate comprising a top electrode;

[0342] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0343] a voltage source operably coupled to the matrix electrodes.

[0344] An electrokinetic device is described herein, comprising:

[0345] a first substrate having a matrix of electrodes, wherein each matrix electrode is coupled with a thin film transistor, and wherein the matrix electrodes are externally coated with a functional coating comprising:

[0346] one or more dielectric layers in contact with the matrix electrodes comprising silicon nitride, hafnium oxide, or aluminum oxide,

[0347] a conformal layer in contact with the dielectric layer comprising parylene, and

[0348] a hydrophobic layer in contact with the conformal layer;

[0349] a second substrate comprising a top electrode;

[0350] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0351] a voltage source operably coupled to the matrix electrodes.

[0352] The electrokinetic device can be used with other elements, such as for example a device for heating and cooling the device or a reagent cartridge for introducing reagents as needed.

[0353] A "droplet" refers to a volume of liquid that is electro-wettingly hydrophobic and is at least partially bounded by a carrier liquid and / or in some cases by a gas or gas mixture, such as ambient air. For example, a droplet can be completely surrounded by a carrier liquid, or can be bounded by a carrier liquid and one or more surfaces of an EWoD device. Droplets can take a wide variety of shapes; non-limiting examples include generally include disc, slug shaped, spheroid, ellipsoid, sphere, partially compressed sphere, hemisphere, oval, cylinder, as well as various shapes formed during droplet operations, such as merging or splitting processes, or due to contact of such shapes with one or more working surfaces of an EWoD device. Droplets can comprise a typical polar fluid, such as water, as in the case of aqueous or non-aqueous compositions, or can be a mixture or emulsion comprising aqueous and non-aqueous components. Droplets can also comprise dispersions and suspensions, for example magnetic beads in an aqueous solvent. Droplets can also comprise one or more surfactants. In various embodiments, droplets can comprise biological samples, such as whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, single or multi-cell containing liquids, organelle containing liquids, fluidized tissues, fluidized organisms, multi-cell organism containing liquids, biological swabs, and biological washes.

[0354] "Droplet manipulation" refers to any manipulation of one or more droplets on a microfluidic device. Droplet manipulation can include, for example: loading a droplet into a 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 place; incubating a droplet; heating a droplet; evaporating a droplet; cooling a droplet; processing a droplet; transporting a droplet out of a microfluidic device; other droplet manipulations 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 will be appreciated that when such terms are used in reference to two or more droplets, any combination of droplet manipulations sufficient to cause the two or more droplets to combine into one droplet can be used. For example, "merging droplet A with droplet B" can be accomplished by transporting droplet A into contact with stationary droplet B, transporting droplet B into contact with stationary droplet A, or transporting droplet A and droplet B into contact with each other. The terms "splitting," "separating," and "dividing" are not intended to imply any particular outcome with respect to the volumes of the resulting droplets (i.e., the volumes of the resulting droplets can be the same or different) or the number of resulting droplets (the number of resulting droplets can be 2, 3, 4, 5, or more). The term "mixing" refers to a droplet manipulation that results in a more uniform distribution of one or more components within a droplet. Examples of "loading" droplet manipulations include, but are not limited to, microdialysis loading, pressure-assisted loading, robotic loading, passive loading, and pipette loading. Droplet manipulations can be electrode-mediated. In some cases, droplet manipulations are further facilitated by the use of hydrophilic and / or hydrophobic regions on a surface and / or by physical barriers.

[0355] For example, 24 DNA constructs can be screened under 8 different conditions to determine the best construct and expression conditions. The 24 constructs can be 24 independent sequences for different POIs, a single POI with 24 different flanking (solubility tags, etc.), or a combination thereof (e.g., 2 POIs with 12 flanking, 3 POIs with 8 flanking, 4 POIs with 6 flanking, etc.). The 8 conditions can be lysates or recombinant expression systems with different components or additives. 192 conditions can be screened in parallel. After the best expression system for the selected POI is identified, reagents / droplets can be selected for purification screening. For example, 30 liquid volumes (droplets) can be selected for screening, and then beads are added to them. By repeatedly merging and splitting with other droplets containing beads, the bead volume can be made uniform for even distribution of beads among droplets. The uniform bead droplets are then merged with droplets containing expressed protein selected for purification studies. The beads can be washed one or more times to remove unbound material. The POI can be eluted from the beads. Once a detection reagent is added to the eluted POI, the soluble material can be detected. Absence of signal indicates that the POI was not bound to the beads or eluted from the beads. If a detectable signal is seen as a clump or patch, it indicates that the eluted POI has aggregated during purification / elution, which is not a good candidate for scale-up. The condition where a high level of uniform signal is seen is selected, and the same determined expression and purification conditions are used for subsequent scale-up. Using the required volume of reagents, the condition that provides the best purification can be scaled up in a test tube outside the device to achieve a sufficient amount of soluble protein. The signal level measured by the purification screen can determine the correct scale-up volume to achieve the required mass of the selected protein. Such scale-up can achieve, for example, more than 20 μg of protein in a 100 μL volume. At the appropriate scale, milligram quantities of protein can be obtained depending on the expression level of the construct and the volume of solution used. Construct screening with different flanking sequences allows placement of, for example, solubility tags at the C- and / or N-terminus and changing the configuration of binding and detection tags, thereby increasing the likelihood that a particular POI can be expressed and purified in soluble form.

[0356] Experiments and Results

[0357] Figure 5 Experimental results for 24 different proteins expressed in a reconstituted cell-free protein synthesis system in droplets on an electrowetting on dielectric (EWoD) device. Each construct contained a GFP 11 tag. In the row labeled "Screening," the GFP 1-10 detection material was present from the start of expression. The row labeled "End Point" shows the absence of GFP 1-10The material was detected for 10 hours, then the GFP 1-10 The fluorescent signal of the material was detected for 5 hours. This experiment shows that there is a significant difference between expression / complementation in the "screening bio-ink" compared to "end-point" detection. The detection of protein clusters formed after expression with end-point detection only implies that the protein aggregates after expression, thus reducing the soluble yield. It is evident from the images that several constructs have spots, indicating that protein aggregates are likely present. The level of aggregation allows the identification of conditions worth further testing and the identification of conditions with high levels of aggregation, from which it is not possible to obtain material further purified.

[0358] VEGF expression

[0359] Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in tumor growth and metastatic spread. VEGF is also implicated in neovascular age-related macular degeneration and rheumatoid arthritis.

[0360] Previously, yeast, insect, and mammalian cell expression systems have been utilized to recombinantly produce VEGF protein in vivo. Due to the intrinsic cysteine knot motif in the human VEGF protein, expression of this protein in E. coli has proven challenging and can lead to protein misfolding and the formation of inclusion bodies. Expression of soluble VEGF in E. coli is only possible in the presence of a solubility fusion tag, such as maltose binding protein (MBP). Furthermore, MBP-VEGF protein can only be successfully expressed in the E. coli Origami 2 (CE3) strain, which carries specific genetic mutations that promote the correct formation of disulfide bonds. Optimizing protein expression in E. coli cells can be both laborious and time-consuming, requiring days to weeks to find the ideal conditions for expressing and purifying soluble protein. E. coli protein expression involves cloning the protein of interest into various expression plasmids encoding different solubility tags, attempting expression in different E. coli cell lines, and optimizing different expression conditions. The inventors have utilized cell-free reagents derived from E. coli to produce VEGF in a microfluidic system.

[0361] Figure 8 Expression screening of VEGF proteins with different flanking sequences was shown. The eight protein constructs generated were linked to different solubility tags at the N- or C-terminus. For purification purposes, a Strep II tag was added (Table 1):

[0362] Table 1. DNA constructs corresponding to VEGF with different solubility tags at the N- and C-terminus.

[0363]

[0364] First, expression and solubility screening was performed to determine optimal expression of human VEGF. This screening used three different cell-free reagents combined with constructs containing eight different nucleic acid template variants corresponding to VEGF with different solubility tags at the N- and C-termini. These three cell-free mixtures consisted of different cell-free protein synthesis reagents. Mixture 3 was designed to promote disulfide bond formation, while Mixture 4 contained a molecular chaperone that promotes protein folding. Mixture 2 contained the core components of the cell-free synthesis reagent and contained no other components. Each construct was run twice. A total of 192 droplets were run on a single cassette. Dashed lines indicate cell-free mixtures run with the corresponding constructs. The location of the detection reagent (DET) is also labeled. Bright white fluorescent spots indicate the expression of soluble proteins.

[0365] Figure 9 show Figure 8 Graphical representation of spot intensity. The concentration of expressed protein is calculated based on the measured fluorescence intensity. Construct A5 (SUMO_VEGF_STREP_DET) produced the highest expression in Mixture 3. The construct with the highest yield (mg / mL) was selected for on-cassette purification (highlighted by the green dashed box).

[0366] Figure 10 Images of 30 soluble candidates are displayed. Candidates exhibiting the highest expression yield are automatically selected and proceed to the on-cassette purification step. These candidates include SUMO-VEGF expressed in mixtures 2, 3, and 4. These expression candidates are purified on-cassette using magnetic beads. Figure 5 The location of the purified SUMO_VEGF_STREP_DET on the cassette is shown. Fluorescence intensity measurements are used to determine the yield of the purified protein.

[0367] Figure 11 show Figure 10 The results are presented graphically, showing a side-by-side comparison of expression and purification yields of SUMO-VEGF-STREP_DET. The results show that the construct was expressed in all three cell-free mixtures, was soluble, and was purifiable. Mixture 3 produced the highest protein expression and purification yields and was therefore selected for cassette-free, scale-up expression.

[0368] Add the linear DNA construct corresponding to SUMO_VEGF_STREP_DET to a test tube containing 500 µL of Mixture 3 expression reagent and incubate overnight. Purify the expressed protein using magnetic beads. Elute the Sumo-labeled VEGF protein with 100 µL of elution buffer, as follows: Figure 12SDS-PAGE gel analysis seen that the eluted protein was >95% pure. The final purified yield of SUMO_VEGF was 32.1 pg of protein. Figure 12 Gel showing the off cassette, scale up expression (500 pL CFPs reaction) and purification of SUMO_VEGF_STREP_DET. SDS-PAGE shows that the VEGF protein has a slightly higher molecular weight than the predicted molecular weight of 36.32 kDa, as SUMO tags typically exhibit higher than their calculated molecular weight on SDS-PAGE gels. Lane 1 molecular weight standard, Lane 2 Mixture 3 negative control, Lane 3 total CFPs after expression, Lane 4 CFPs obtained after centrifugation (soluble) and soluble fraction obtained, Lane 5 unbound; flow through sample that did not bind to the beads, Lane 6 purified (eluted); sample eluted from the resin beads.

[0369] VEGF protein validation data summary and conclusions

[0370] VEGF is a difficult candidate to express in an E. coli expression system. Un-tagged VEGF tends to form inclusion bodies at the end of expression. To obtain soluble protein, VEGF was expressed with a variety of solubility tag combinations to determine the correct construct for successful expression and purification. Given the diversity of constructs that needed to be tested, traditional cell-based expression and purification methods were time consuming and labor intensive. We have demonstrated that the droplet microfluidic platform enables rapid construct expression screening and selection of soluble, purifiable candidates. VEGF with an N-terminal SUMO tag gave the highest expression yield in Mixture 3 expression reagents. The results generated by the platform were validated by off-cassette cell-free expression. The expressed protein was purified to high purity and high yield; comparable to VEGF protein produced by cell-based expression platforms.

[0371] Uniformization of beads in 8x4 array and repositioning of droplets after each cycle

[0372] Magnetic particles were visualized and quantified by fluorescence imaging of fluorescein bound to the surface of the magnetic particles. A 32x array of droplets containing 25 pm streptavidin-coated magnetic particles was dispensed into 49 px 2 droplets in an 8x4 format. Figure 13 Repeats of four arrays on two devices are shown.

[0373] As Figure 14 shown, each array was subjected to several cycles (40 cycles in this case) of repeated mixing and splitting of droplets containing magnetic beads, as per the flow diagram below. Figure 15 Fluorescence images of the arrays after cycling are shown. Figure 13 and Figure 15 Comparison of Figure 13There is a significant difference between the brightest and darkest droplets in the starting image in Figure 1, indicating a large difference in bead concentration. Figure 15 The display shows a much lower level of droplet brightness variation, indicating that the bead concentration has become uniform.

[0374] The fluorescence signal intensity of each droplet was analyzed to compare the bead mass of each droplet before and after cycling:

[0375]

[0376] The data show that bead concentration can be normalized by repeatedly combining and splitting droplets, then moving to the next droplet and combining and splitting. Each droplet is mixed with at least one other droplet to allow diffusion of beads between droplets and to overcome any initial errors due to bead partitioning volume.

[0377] Uniformization of beads measured by clumping and resuspension of uniform droplets.

[0378] The 4x8 array homogenization as described above was performed on the same cartridge as the bead array that was not homogenized, this was performed on a 4x cartridge, each cartridge containing 32 homogenized droplets and 32 non-homogenized droplets. The un-mixed mixed beads lost 21.1% of the bead population (27 out of 128), while the homogenized / mixed beads lost <1% of the bead population (1 out of 128). Figure 17 The display shows the size distribution of the population. In the various droplets, the beads are mixed uniformly, and the number of droplets that completely lose beads is greatly reduced.

Claims

1. A method for generating aqueous droplets having a uniform concentration of suspended beads, wherein the method comprises: a. A container for taking out an aqueous liquid containing beads suspended within it. b. Forming multiple droplet volumes from the reservoir. c. Merge two or more droplets. d. Droplets splitting and merging, e. Rearranging the droplet assembly, and f. Repeat steps c, d, and e once or more, so that each final split droplet contains a uniform concentration of suspended beads.

2. The method according to claim 1, wherein the beads are magnetic.

3. The method according to claim 1 or claim 2, wherein each droplet formed from the reservoir merges and splits with each other droplet formed from the reservoir.

4. The method according to any one of claims 1 to 3, wherein eight or more droplets are generated.

5. The method according to any one of claims 1 to 3, wherein 32 or more droplets are generated.

6. The method according to any one of claims 1 to 5, wherein the method is performed in a droplet on a digital microfluidic device.

7. The method of claim 6, wherein the digital microfluidic device comprises an active matrix thin-film transistor.

8. The method according to any of the preceding claims, wherein the diameter of the beads is greater than 10 μm.

9. The method according to any of the preceding claims, wherein the average diameter of the beads is 25 μm.

10. The method according to any of the preceding claims, wherein uniform bead droplets are merged with droplets having a substance bound to the beads.

11. The method according to any of the preceding claims, wherein the beads are used to bind to a protein.

12. A method for synthesizing, characterizing, and purifying one or more proteins having a detection tag and a binding tag, the method comprising the steps of: i. Mix the cell-free protein expression system with one or more nucleic acid templates to form multiple combined liquid volumes; iia divides the merged volume into at least two equal portions and achieves protein expression in at least two equal portions; or iib is used to express the protein, and then the sample is split into at least two equal portions. iii. Add a detection reagent bound to the detection tag to at least one aliquot of the sample to determine the protein expression level and the conditions under which the protein is expressed in a soluble form; iv Select a volume in which the protein has been solublely expressed and add magnetic beads to a subset of the aliquot sample, wherein the beads are bound to a binding tag and the beads in the droplets have been merged and split once or multiple times, such that each final split droplet contains a uniform concentration of suspended beads. v. Separate the magnetic beads from the expression reagent; vi Optionally, a detergent is added to the magnetic beads to remove unbonded material; vii. The bound protein is eluted from the magnetic beads by disrupting the binding to the binding tag; as well as viii. Add a detection reagent bound to a detection tag to at least one eluted aliquot to determine the level of protein purification and the conditions for obtaining purified soluble protein.

13. The method of claim 12, wherein the ratio of soluble target protein to insoluble target protein is determined in the volume of the added detection reagent.

14. The method according to any one of claims 12 or 13, wherein each binding sequence comprises four or more amino acids.

15. The method of claim 14, wherein the detection reagent comprises a component of a fluorescent protein.

16. The method of claim 15, wherein the target protein has ccGFP. 11 The label and the detection reagent contain ccGFP. 1-10 .

17. The method according to any one of claims 12 to 16, wherein the POI is expressed together with a solubility enhancer selected from: 。 18. The method according to any one of claims 12 to 17, wherein the expression is performed using a cell-free lysate or a reconstructed expression system.

19. The method according to any one of claims 1 to 18, wherein the digital microfluidic device comprises an oil-filled environment or a humidified gas environment, wherein the humidified gas environment is achieved by enclosing or sealing the digital microfluidic device and providing an onboard reagent reservoir.

20. The method according to any one of claims 1 to 19, wherein the beads are bonded to a binding portion selected from: α tag (SRLEEELRRRLTE) Avi tags (GLNDIFEAQKIEWHE) C-label (EPEA) Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) Dogtag(DIPATYEFTDGKHYITNEPIPPK) E-label (GAPVPYPDPLEPR) FLAG (DYKDDDDK) G4T (EELLSKNYHLENEVARLKK) HA (YPYDVPDYA) His (HHHHHH) Isopeptide tag (TDKDMTITFTNKKDAE) Lanthanide binding tags (LBT) (FIDTNNDGWIEGDELLLEEG) Myc (EQKLISEEDL) NE tag (TKENPRSNQEESYDDNES) Polyglutamic acid tag (EEEEEEE) Polyarginine tag (RRRRRRR) Rho1D4 tag (TETSQVAPA) SBP label (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) Sdytag (DPIVMIDNDKPIT) SH3 (STVPVAPPRRRRG) SNAC (GSHHW) Snooptag (KLGDIEFIKVNK) Softag 1 (SLAELLNAGLGGS) Softag 3 (TQDPSRVG) Spot label (PDRVRAVSHWSS) Spytag (AHIVMVDAYKPTK) S-label (KETAAAKFERQHMDS) Strep tag (AWAHPQPGG) (AWRHPQFGG) Strep Tag II (WSHPQFEK) T7tag (MASMTGGQQMG) TC label (EVHTNQDPLD) Ty tag (CCPGCC) VSV tag (YTDIEMNRLGK) Xpress Tag (DLYDDDDK).

21. The method according to any one of the preceding claims, comprising: One or more proteins are expressed in one or more droplets on a digital microfluidic device having a two-dimensional planar microelectrode array, wherein the proteins have ccGFP. 11 The peptide amino sequence tag and the Strep tag split the droplet into at least two volumes, which will contain ccGFP after the expression process. 1-10 The droplets were merged into one droplet, and the uniformity of the fluorescence signal was measured to determine the degree of aggregation of the expressed protein. A sample containing the already soluble expressed protein but without ccGFP was taken. 1-10 The corresponding droplets are added, and magnetic beads are attached to the strep label, wherein the droplets containing magnetic beads have been merged and split once or multiple times, such that each finally split droplet contains a uniform concentration of suspended beads, the beads are fixed and washed to remove unbound material. The POI was eluted by disrupting strep binding, followed by the addition of ccGFP. 1-10 The droplets were analyzed, and the fluorescence signal was measured to determine the level of protein purification and the conditions for obtaining purified soluble protein.

22. The method according to any of the preceding claims, wherein at least eight different nucleic acid templates are screened for at least four different expression reagents on the same apparatus.

23. The method according to any of the preceding claims, wherein the device is capable of manipulating at least 32 droplets containing beads.

24. The method according to any of the preceding claims, wherein the final distribution of the beads has a uniform concentration of 20% v / v+ / -10% per droplet.

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