Protein aggregation assays

Abstract

The invention provided herein relates to methods for measuring protein aggregation and stability. The method can be used for characterizing cell-free protein synthesis reactions.

Description

Field of the Invention

[0001] Provided herein are methods for improving the expression and characterization of proteins in cell-free systems.

[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 a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA, or RNA. There are various protein purification strategies to address the desired scale, yield, and downstream application issues. However, protein production can be challenging for many reasons. A major challenge is finding a suitable expression system, such as one derived from mammalian, bacterial, fungal, or plant cells. This can require 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 the use of living cells. The CFPS environment is not limited to the cell wall or the conditions required to maintain cell viability, and can rapidly produce any desired protein from a nucleic acid template, typically plasmid DNA or RNA from in vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis includes systems based on crude lysates (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275–284). CFPS requires high concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, etc. (Febs Letters 2013, 2, 58, 261-268).

[0005] Split-type detectors, such as the split green fluorescent protein (GFP) system, are known for use in protein expression. A target protein containing a GFP subcomponent can be detected by complementation with a detection agent containing the remaining GFP portion. Cabantous and Waldo described such a system (In Vivo and In Vitro Protein Solubility Assays Using Split-Type GFP (NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 845)). This system relies on expressing the target protein in cells, cleaving it from the cells, and then exposing it to a detection agent to measure expression levels.

[0006] WO2022 / 038353 describes the cell-free expression of a GFP-tagged protein in the presence of a GFP detection substance to measure the signal during expression.

[0007] Many proteins containing endogenous soluble factors have been expressed. However, factors such as maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), glutathione S-transferase (GST), or thioredoxin (TRX) have considerable size and may have undesirable effects on the target protein to which they are attached. In addition, the expression of large solubility elements uses reagent sources during expression. Such sequences also take time to fold before acting as solubility enhancers, so synthesizing a target protein with a solubility-enhancing sequence attached to it is far from ideal. Applicants herein wish to produce proteins without the need for attached soluble factors.

[0008] To date, protein purification and analysis have generally required complex analytical techniques, including electrophoresis, or the need for purified proteins. The inventors herein have developed simplified protein analysis methods that enable characterization of expressed proteins in crude form in a variety of ways.

[0009] WO2007112677 and CN112646826 disclose the use of fusion proteins, such as Trx, for expression. Trx is a common protein found in yeast, bacteria, animals, and plants, and is also endogenous to commonly used hPTH(1-34) expression hosts, such as yeast and Escherichia coli. This protein can regulate the balance between protein folding and aggregation in cells. Trx can interact with many proteins and enhance the solubility of fusion proteins, thereby reducing inclusion body formation. Fusion proteins consist of Trx linked to the target protein.

[0010] US2006099710 discloses the expression of an MBP fusion protein, wherein the MBP enhances the solubility of the expressed protein. The vector contains a sequence that expresses a protease cleavage site (TMV protease). Expression occurs, and the MBP is cleaved. The MBP fusion enhances the solubility of the expressed protein by being linked to the target protein's sequence.

[0011] KR101828672 describes an introduction vector containing a codon-optimized, synthesized DNA sequence encoding the 165-amino acid interferon α2b. Seven tags (His6, Trx, GST, hPDIb'a', MBP, hPDI, and NusA) are fused to the N-terminus of IFNα2b. Tobacco etch virus recognition sites (TEVrs; ENLYFQ↓G) for removal of tagged proteins are placed between the tag and IFNα2b in each construct. As described above, the soluble tag is directly linked to the target protein in a continuous sequence.

[0012] WO2018076008 again discloses a fusion protein with a soluble tag linked to a target protein.

[0013] US2022275027 again discloses a fusion protein with a signal peptide linked to a target protein.

[0014] CN108117599 again discloses a fusion protein comprising a chaperone and a toxin polypeptide Ssm6a (also referred to as the "fusion protein of the present invention"), wherein the molecular chaperone promotes the correct folding of disulfide bonds in the toxin polypeptide.

[0015] WO2022038353 describes the use of split fluorescent proteins to detect protein expression, but not as a measure for aggregation.

[0016] WO2014132053 describes a microdroplet assay for protein aggregation, but not as an assay for protein expression.

[0017] One of the current challenges in cell-free protein synthesis is to increase the soluble yield of expressed proteins and avoid aggregation or insolubility. SUMMARY OF THE INVENTION

[0019] The present invention relates to a method for determining the degree of aggregation of a protein in one or more droplets on a digital microfluidics device, wherein the degree of aggregation is measured using optical methods. In one aspect, the present invention relates to determining the degree of aggregation of a protein expressed in a cell-free system, comprising expressing a protein of interest (POI) in droplets on a digital microfluidics device, measuring the expressed POI using a detectable signal, and measuring the level of insoluble POI. The level of aggregation can be used to determine whether the protein is expressed in a soluble or insoluble form. Even if a protein is expressed soluble, it does not necessarily mean that the protein will remain soluble. By observing the aggregation state, the "aggregation propensity" or "stability" of the initially soluble protein can be understood. Soluble proteins can be further purified, while highly aggregated proteins are less likely to be purified or active.

[0020] The protein for screening can be expressed on the device, or can be placed on the device from an external sample, and its source can be a pure protein or obtained via expression in the tube (outside the device). The protein can be used for stability screening, for example, for identifying the buffer and surfactant that the protein is more or less easily aggregated. For example, a protein sample can be screened for a group of buffer and surfactant (i.e. 24 kinds of buffers and 8 different surfactants) to identify the more stable reagent of the protein therein. In such a case, the method is selected to exclude aggregates in highly parallel buffer screening, looking for the best buffer. The rate at which aggregates form can be used to estimate the stability of the protein under different conditions.

[0021] Aggregation can be measured directly, for example using light scattering, or can be observed using a visible marker. Detection can be visualized as optically detectable aggregates that cause light scattering, or detection can produce aggregates that can be detected by labeling, for example using a fluorescent marker. Light scattering can be measured at an angle different from that of 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.

[0022] The degree of aggregation can be measured by counting the number of aggregates, the area of ​​the aggregates, the intensity of the aggregates, or by using turbidity or dispersion measurements. The degree of aggregation may be zero, indicating that the droplet contents are uniform and remain in solution. In such a case, the light signal or fluorescent signal is evenly distributed throughout the droplet, without clustering or agglomeration. Alternatively, the aggregation may be complete, so that only clustered aggregates are detected and the remaining droplets do not produce a signal. Based on the ratio between these two extremes, the signal may be partially aggregated and partially dispersed throughout the droplet.

[0023] The POI may be expressed with or contain a binding sequence that binds to a detection moiety. In this case, additional droplets containing the detection moiety may be added to the droplet containing the POI to generate a detectable signal in the combined droplet.

[0024] The binding sequence may contain four or more amino acids. The binding sequence may contain 4-30 amino acids.

[0025] The detection moiety can be a protein. The detection moiety can comprise a component of a fluorescent protein. The fluorescent protein can contain an additional solubility enhancer, for example, selected from the group consisting of:

[0026] Glutathione S-transferase GST small ubiquitin-like modifier protein SUMO Maltose binding protein MBP Fasciola hepatica 8 kDa antigen FH8 Thioredoxin TRX Solubility-enhanced ubiquitous tags SNUT 17,000 Dalton protein SKP Monomeric bacteriophage T7 orc protein MOCR Escherichia coli secreted protein A ESPA N Utilization of substances NusA Protein G IgG domain B1 GB1 Protein A IgG repeat domain ZZ ZZ Mutated dehalogenase Halo Tags Bacteriophage T7 protein kinase T7PK Escherichia coli trypsin inhibitor Ecotin calcium-binding protein CaBP Stress response arsenate reductase ArsC N-terminal fragment of translation initiation factor IF2 IF2-domain 1 Stress response proteins RpoA Stress response proteins SlyD Stress response proteins Tsf Stress response proteins RpoS Stress response proteins PotD Stress response proteins Crr Escherichia coli acidic protein msyB Escherichia coli acidic protein yjD Escherichia coli acidic protein rpoD T7 phage tail P17 Metal-binding proteins CUSF 53 amino acid long N-terminal extension sequence NEXT

[0027] The tag can be, for example, GFP 11 After POI expression, once the added GFP 1-10, forming a fluorescent complex GFP 1-11 .

[0028] GFP 11 With sufficient length to GFP 1-10 Combined to produce stable binding and from GFP 1-11 Fluorescent signal of GFP. 11 The peptide amino acid sequence tag may be selected from:

[0029] KRDHMVLLEFVTAAGITGT

[0030] KRDHMVLHEFVTAAGITGT

[0031] KRDHMVLHESVNAAGIT

[0032] RDHMVLHEYVNAAGIT

[0033] GDAVQIQEHAVAKYFTV

[0034] GDTVQLQEHAVAKYFTV

[0035] GETIQLQEHAVAKYFTE.

[0036] If desired, the tag can be cleaved from the POI to remove the tag / detection agent. Cleavage can be performed, for example, using a metal cation-based protease.

[0037] The expression of the expressed protein can be determined, for example, using optical measurements without purification from the expression system. Expression and / or stability can be determined at a fixed time or temperature. For example, an unpurified protein is exposed to a set temperature and the sample is analyzed for the presence of soluble POI remaining over time, or an unpurified protein is exposed to a range of temperatures and the sample is analyzed for the presence of soluble POI remaining.

[0038] Stability can be determined by expressing a protein of interest (POI), dividing the unpurified expression product into fractions, exposing at least a portion of the unpurified protein to an elevated temperature, and analyzing the heated sample for the presence of soluble POI remaining when compared to the unheated portion of the sample.

[0039] Stability can be determined by taking a protein of interest (POI), dividing the protein sample into multiple portions, exposing at least a portion of the protein to an elevated temperature, and analyzing the heated sample for the presence of soluble POI remaining when compared to an unheated portion of the sample.

[0040] Stability can be determined by taking a protein of interest (POI), dividing the protein sample into multiple fractions, combining additives with the protein droplets to create different chemical compositions, such as different buffers and surfactants, and analyzing the different compositions for the presence of remaining soluble POI when compared to an untreated fraction of the sample.

[0041] In case of producing amino acid sequence by expression, activity just depends on correct folding.Solubility and activity depend on the stability of the protein expressed.Incorrectly folded protein can be more unstable than correctly folded protein.Therefore, the stability of the protein expressed under test conditions is an important measure of the existence of correctly folded protein.After protein expression, the system of rapid characterization of gained protein can be used to determine the stability of the material expressed, without purifying this material.

[0042] Protein stability shows that the ability of protein to keep correctly folded and / or not to aggregate over time.Stability depends on conditions, such as protein concentration, pH, buffer composition or temperature.Can determine each group of variable conditions, such as thermostability, aggregation tendency, pH stability etc..When determining activity in further determination, it is important to measure the stability of the expressed protein, in order to ensure that the measured property belongs to the protein of expectation folding, rather than oligomer, aggregate or other misfolded structures.

[0043] Assays for protein stability may include changes in solubility, aggregation, thermal stability after thermal excursion, or use of known binding ligands.

[0044] The method can be used to characterize the initial soluble yield of the expressed protein and the stability of the expressed protein. The method can identify the conditions that are optimal for both expression and stability of the expressed protein.

[0045] Aggregation can be measured directly or by adding reagents that detect aggregation. For example, thioflavin is used. Thioflavin is a fluorescent dye, and at least two compounds are available: thioflavin T and thioflavin S. Both are used for histological staining and biophysical studies of protein aggregation, such as for studying amyloid formation. Thioflavin T has been used in the study of Alzheimer's disease and other neurodegenerative diseases.

[0046] Stability can be determined using dynamic light scattering (DLS) of aggregated protein clusters. Stability can be determined using differential scanning fluorimetry (DSF). Stability can be determined by taking time points at a fixed temperature as an isothermal stability test. Alternatively, stability can be measured by heating to different temperatures for a fixed time. Stability can be measured by heating the expressed protein to, for example, greater than 40°C or greater than 50°C.

[0047] Stability can be measured by analyzing the soluble fraction remaining after the heating step or the level of aggregation remaining.

[0048] Stability can be determined over a period of time and compared to initial measurements. The method can include expressing a protein of interest (POI), monitoring the amount of soluble expression, exposing the unpurified protein to a set temperature, and analyzing the sample for the presence of remaining soluble POI over time.

[0049] The method may comprise expressing a protein of interest (POI), monitoring the amount of soluble expression, exposing the unpurified protein to a range of temperatures, and analyzing the samples for the presence of remaining soluble POI.

[0050] The method may include expressing a protein of interest (POI), dividing an unpurified expression product into a plurality of fractions, exposing at least a portion of the unpurified protein to an elevated temperature, and analyzing the heated sample for the presence of remaining soluble POI when compared to an unheated portion of the sample.

[0051] The method may comprise expressing a protein of interest (POI) in a plurality of separate volumes of liquid, exposing at least one volume of unpurified protein to an elevated temperature, and analyzing the heated volume for the presence of remaining soluble POI when compared to an unheated volume.

[0052] 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 a reconstitution system comprising assembled components for transcription and translation in a Purified Recombinant Elements (PURE) system.

[0053] Any of the methods described herein can be performed in, for example, microtiter plates or microcentrifuge tubes. The expressed protein can be placed in a device for high-throughput stability screening. Any of the methods can be performed on a microfluidic or digital microfluidic device. The digital microfluidic device can include an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by sealing or sealing the digital microfluidic device and providing a reagent reservoir for carrying.

[0054] A method for expressing proteins in droplets on a digital microfluidics device having a two-dimensional array of planar microelectrodes is disclosed, wherein the proteins have GFP 11 Peptide amino acid sequence tag, where a portion of the droplets contain GFP during the expression process 1-10 , and the other part has GFP added after expression 1-10 , and compared GFP from the droplets 1-11 The level of the signal.

[0055] Expression comparisons can identify optimal conditions for expressing the desired protein in the most soluble and stable form.

[0056] Attached photos

[0057] Figure 1 The target protein with a peptide tag (detection tag) is displayed in the presence of a protein bound to the peptide tag (detection protein). If the detection tag and the detection protein produce a detectable signal upon binding, the amount of the target protein can be measured. Additionally or alternatively, if the detection protein is a solubilizing factor, it can increase the solubility of the target protein after binding to the peptide tag. The solubilizing ability of such a binding system can be superior to that of a soluble factor of the fusion protein.

[0058] Figure 2 Animated illustration showing protein expression of different proteins with varying soluble yields and aggregation levels. Some conditions show no expression (dark color). Some conditions produce high levels of soluble protein (white squares of uniform intensity). Some conditions produce protein aggregates, meaning the expressed protein is insoluble and clumps together (white dots on a dark background). Some conditions produce a mixture of soluble and aggregated protein (white dots on a gray background). Therefore, soluble expression levels and aggregation levels can be determined by adding a test substance to the expressed protein.

[0059] Figure 3 A schematic workflow for protein expression is shown. A DNA linear expression construct (LEC) can be mixed with expression reagents and combined. The mixed reaction can be divided into multiple aliquots and subjected to protein expression. After expression occurs, a soluble detection substance (DET) is added to one drop. When the expressed protein is soluble and has low aggregation, the signal is uniform, indicating soluble expression.

[0060] Figure 4 A schematic workflow for protein expression is shown. A DNA linear expression construct (LEC) can be mixed with expression reagents and combined. The mixed reaction can be divided into multiple aliquots and subjected to protein expression. After expression occurs, a soluble detection substance (DET) is added to the droplet. When the expressed protein aggregates, the detection agent gives a nonuniform signal, indicating that the expressed protein is insoluble or aggregated to some extent. Insoluble or aggregated POIs are less likely to be purified from the droplet.

[0061] Figure 5 Shown are experimental results for 24 different proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstituted cell-free protein synthesis system. Each construct contained GFP 11 In the row labeled "Filter," GFP 1-10The test substance is present from the beginning of expression. The row marked "end point" shows the absence of GFP. 1-10 The detection substance was expressed for 10 hours and then GFP was used 1-10 Fluorescence signal was measured after 5 hours of complementation. This experiment revealed significant differences between expression / complementation in the Screen BioInk compared to endpoint detection. The formation of protein clusters following expression, detected only by endpoint detection, suggests that the protein aggregates after expression, reducing soluble yield. Aggregates are evident in several constructs, indicating the high likelihood of protein aggregates. Aggregation levels can identify conditions worthy of further testing and conditions with proteins exhibiting high levels of aggregation, from which further purification would be unlikely to yield material.

[0062] Figure 6-8 Shown are the expression results of two proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstituted cell-free protein synthesis system. Eight different cell-free expression blends were tested for each protein. Figure 6 White light images after 10 h of expression are shown. Figure 7 White light images after addition of the test protein (another 5 h) are shown. Figure 8 Fluorescence (blue light) images after adding the test protein are shown. For the soluble protein (DHFR) (left side of the array), the droplets appear uniform in all three images because the expressed protein does not appear in clumps, with no aggregation or clumping observed in any of the three images. In the white light images, the droplets exhibit varying levels of turbidity, depending on the expression level. In the central three columns of the images after expression ( Figure 6 ), protein clusters (P17_IFN) can be detected in the optical image. After adding the detection protein, clusters can still be detected in the white light image ( Figure 7 ). Fluorescence image ( Figure 8 ) shows fluorescent clusters within the droplets, indicating the presence of protein aggregates. Droplets with high levels of turbidity in the white light image also have high levels of signal from bound detection protein, demonstrating that the protein can be detected using either white light or fluorescence measurements. Detailed Description of the Invention

[0064] Applicants are able to visualize protein aggregates directly in droplets on a microfluidic device. Such visualization methods enable direct visualization of protein stability using optical readouts. Applicants have recognized that the level of aggregation after protein expression is an important measure for determining whether the protein can subsequently be purified. High levels of protein aggregation indicate a lack of soluble protein for purification. In such cases, the protein is expressed, but aggregates or misfolds after expression, in contrast to proteins that fail to express the correct sequence. Disclosed herein is a method for determining the degree of aggregation of proteins expressed in a cell-free system, comprising expressing a protein of interest (POI) in droplets on a digital microfluidic device, wherein the expressed POI is measured by an optically detectable signal, and the level of insoluble POI is measured.

[0065] Further disclosed is a method for protein synthesis comprising expressing a protein of interest (POI) and measuring the stability of the expressed protein using optical measurements without purification from the expression system.

[0066] When expressing proteins, various parameters are important in determining successful expression, both in terms of yield and functionality. One factor is the soluble yield of the expressed protein, as many proteins are expressed in an insoluble form or become inactive due to a lack of stability.

[0067] The soluble yield assay described herein involves expressing and detecting the expressed protein to measure the degree of aggregation. The protein of interest (POI) can have a binding tag that binds to a detection agent to generate a signal. The detection agent can also have additional soluble factors.

[0068] The method can be used as part of a process to determine whether an expressed protein is suitable for subsequent purification testing. For example, the method may include expressing a protein of interest (POI) in a first reagent volume and dividing the reagent volume into multiple aliquots. The protein of interest may have a binding tag that is complementary to a detection substance and becomes fluorescent. The detection substance is added to one or more aliquots, and the signal is recorded. The uniformity of the signal within the droplet determines the degree of aggregation. When the signal is uniform within the droplet, the POI is soluble and exhibits little or no aggregation. When the signal is heterogeneous within the droplet, visible as clumps, the protein is aggregated and insoluble. Therefore, the overall signal level indicates expression level, while signal uniformity determines aggregation. Therefore, expression level and soluble expression level can be determined by observing the amount and uniformity of the signal within the droplet. The degree of aggregation can be measured using uniformity measurements, counting the number of aggregates, their area, or their intensity, or using dispersion measurements.

[0069] If the protein is unstable or poorly soluble, the protein may aggregate. Protein aggregates result in a lack of uniform signal in white light optical images, or this phenomenon occurs from the addition of a detection agent. Therefore, POIs with high soluble yields produce uniform levels of signal, while proteins with low soluble yields can have low or high levels of signal, but because the protein is aggregated, the signal aggregates and is uneven throughout the reaction volume. Aggregated proteins are likely to be less desirable for subsequent determination or purification.

[0070] Further protein stability assays may include

[0071] Solubility

[0072] Aggregation (over time)

[0073] ●Thermal stability.

[0074] Protein thermal stability does not necessarily predict the aggregation rate at low temperatures (and vice versa). For example, the melting temperature (T) of a monoclonal antibody m ) does not necessarily correlate with the aggregation rate of the monoclonal antibody at room temperature (Mol. Pharmaceutics 2016, 13, 307−319). At high temperatures, the aggregation rate is determined by the unfolded state, while at low temperatures, the aggregation rate is determined by the native state. Therefore, denaturing a protein may or may not provide an accurate measure of the stability of the protein in its native state.

[0075] Providing protein aggregation measurement over time would be very useful, in addition to simply measuring expression yield, also provides other information. Producing stable protein formulations is the key to protein development and manufacturing. Since proteins may be exposed to various types of stress, such as temperature, pH, salt, mechanical stress, surface interaction or oxidation, the robustness of the formulation against external stress factors is crucial. Protein aggregation is a common problem faced during expression and purification. Therefore, it is very necessary to predict or control protein aggregation during the generation of target protein. This paper describes a high-throughput assay for protein aggregation / stability, which uses directly expressed materials to measure protein stability.

[0076] Protein purification can be challenging and time-consuming, often resulting in significant material losses. Therefore, it is desirable to measure protein stability directly after expression without having to purify the protein from the protein used in expression. Measurements in crude cell lysates or directly in cell-free expression systems are possible using optical-based analytical techniques, as these techniques can tolerate the presence of other proteins that are stable to the conditions being measured or that would otherwise be undetectable.

[0077] A method for protein synthesis is disclosed, comprising expressing a protein, wherein the expressed protein contains a subcomponent of a fluorescent protein, and monitoring the stability of the protein over time by producing the fluorescent protein at different times in different batches of material.

[0078] The method avoids the need for complex purification steps or gel-based separations. Fluorescence-based assays can be used to measure the amount of expressed protein (soluble yield) in conjunction with protein stability. Thus, both yield and stability can be determined post-expression without the need for gel running or purification of materials.

[0079] Fluorescence measurements can be used as real-time measurements of soluble protein expression. When the tag sequence is produced, the presence of the remaining fluorescent protein as a detection substance enables real-time measurement of the tag sequence and the amount of soluble protein. Alternatively, fluorescence can be measured by adding a detection substance 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 stabilization step to determine the remaining protein level. Therefore, both the yield of the synthesis and the stability of the expressed protein can be determined. For example, GFP 11 Can be linked to expressed proteins, GFP 1-10 Used as a test substance.

[0080] This screening workflow enables users to rapidly screen different expression systems in the form of cell-free lysates or reconstitution systems. Once the optimal expression system is identified, soluble yield and stability can be measured directly without having to isolate the target protein from a complex mixture containing other proteins, nucleic acids, and other cellular components.

[0081] 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 that is specific for a particular antibody. The tag can be attached to the N-terminus or the C-terminus. For example, the binding moiety can be selected from the following list of exemplary peptide affinity binding sites:

[0082] α-Tag (SRLEEELRRRLTE)

[0083] Avi-Tag (GLNDIFEAQKIEWHE)

[0084] C-label (EPEA)

[0085] Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0086] Dogtag(DIPATYEFTDGKHYITNEPIPPK)

[0087] E-tag (GAPVPYPDPLEPR)

[0088] FLAG (DYKDDDDK)

[0089] G4T (EELLSKNYHLENEVARLKK)

[0090] HA (YPYDVPDYA)

[0091] His (HHHHHH)

[0092] Isopeptide tag (TDKDMTITFTNKKDAE)

[0093] Lanthanide Binding Tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0094] Myc (EQKLISEEDL)

[0095] NE-Tag (TKENPRSNQEESYDDNES)

[0096] Polyglutamic acid tag (EEEEEEE)

[0097] Polyarginine tag (RRRRRRR)

[0098] Rho1D4-Tag (TETSQVAPA)

[0099] SBP-Tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQR EP)

[0100] Sdytag(DPIVMIDNDKPIT)

[0101] SH3 (STVPVAPPRRRRG)

[0102] SNAC (GSHHW)

[0103] Snooptag (KLGDIEFIKVNK)

[0104] Softag 1 (SLAELLNAGLGGS)

[0105] Softag 3 (TQDPSRVG)

[0106] Spot-Tag (PDRVRAVSHWSS)

[0107] Spytag (AHIVMVDAYKPTK)

[0108] S-tag (KETAAAKFERQHMDS)

[0109] Streptavidin-tag (AWAHPQPGG) (AWRHPQFGG)

[0110] Streptavidin-Tag II (WSHPQFEK)

[0111] T7 tag (MASMTGGQQMG)

[0112] TC-Tag (EVHTNQDPLD)

[0113] Ty-Tag (CCPGCC)

[0114] VSV-Tag (YTDIEMNRLGK)

[0115] Xpress-Tag (DLYDDDDK).

[0116] The binding moiety tag can be a subcomponent of the fluorescent protein. Thus, the fully assembled protein becomes fluorescent. For example, if the detection protein contains GFP 1-10 , and the expressed protein tag contains GFP 11 The peptide complements the fluorescent GFP, enabling simultaneous monitoring and stability assessment. By monitoring changes in the levels of aggregated material that produces a fluorescent signal, the expressed material can be monitored over time or under different conditions.

[0117] The binding moiety may comprise a small molecule affinity tag, such as biotin. The binding moiety may comprise a specific nucleic acid sequence.

[0118] The process can be performed in droplets that can be manipulated electrokinetically to enable and improve protein expression and analysis. Any electrokinetic means can be used to move the droplets. Electrowetting on dielectrics (EWoD) can be used to move the droplets. Electrical signals on EWoD or optical EWoD devices can be delivered via segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.

[0119] Cell-free expression of peptides or proteins can use cell lysates or reconstitution systems with reagents for protein expression. Common components of cell-free reactions include energy sources, amino acid supply sources, cofactors such as magnesium, and related enzymes. Cell extracts are obtained by lysing target cells and removing cell walls, DNA genomes, and other fragments by centrifugation. The rest are cellular machinery, including ribosomes, aminoacyl-tRNA synthetases, translation initiation factors and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using an expression machine derived from a cell.

[0120] In order to optimize expression, the expression system can be supplemented with additional components, including purified enzymes. Additional components can include salts, cofactors, buffers, surfactants, partners, or additional protein components. Additional protein components can be selected from, for example, partners, glycosylases, proteases, redox-active enzymes, phosphorylases, and kinases.

[0121] Expression compositions can be assembled on the device starting from mixing various droplets to screen various compositions in parallel.

[0122] As an example, screening reagents may include:

[0123] ■ Mate mixtures (e.g. PUREfrex GroE Mixture )

[0124] ■ Kinase 1 (e.g. NEB CK2 )

[0125] ■ Kinase 2 (e.g. NEB PKA )

[0126] ■ Protease 1 (e.g. NEB TEV )

[0127] ■ Protease 2 (e.g. Merck HRV 3C )

[0128] ■ Common metal ion mixtures

[0129] ■ Common cofactor mixtures.

[0130] The user can blend the compositions and monitor the expression level of the target protein under each blending condition.

[0131] Any specific 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 synthetically. LECs can be prepared via PCR. mRNA can be produced using an in vitro transcription system. 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 containing a different nucleic acid template.

[0132] An energy source is an essential component of cell-free reactions. Typically, a separate mixture containing the required 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 creatine phosphate. During expression, the energy source can be supplemented by adding other reagents to the droplets during the process.

[0133] A cell-free extract with components for protein expression contains everything needed for protein expression except the nucleic acid template. Thus, the term includes all associated ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions, and energy sources. Once the nucleic acid template is added, protein expression begins, and no other reagents are required.

[0134] Therefore, the cell lysate can be supplemented with additional reagents before the template is added. Cell-free extracts containing the components for protein expression will generally be produced as a bulk reagent or 'master mix', which can be prepared into many identical droplets before different templates are added to different droplets. Common cell extracts currently in use are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE), and Kluyveromyces yeast (D2P system). All of these extracts are commercially available.

[0135] Cell-free systems can be assembled from the required reagents, rather than from cell extracts. Systems based on reconstituted, purified molecular reagents are commercially available, such as the PURE system for protein production, 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. The protein synthesis reactions in the PURE system lack proteases and ribonucleases, which are often present as undesirable molecules in cell extracts.

[0136] Using a population of droplets with different compositions allows for rapid screening of various variables to identify optimal conditions for expressing a desired protein. Proteins can also contain sequences with additional amino acid domains, such as solubility factors or binding tags.

[0137] Protein soluble sequences can be linked to detection sequences. For example, GFP 1-10 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 modifier protein (SUMO), glutathione S-transferase (GST), or thioredoxin (TRX). The tag can be linked to the C-terminus or N-terminus. Any example of a solubility enhancer can be used. A list of possible proteins is shown below. Any sequence selected from the following list can be selected:

[0138] Glutathione S-transferase GST small ubiquitin-like modifier protein SUMO Maltose binding protein MBP Fasciola hepatica 8 kDa antigen FH8 Thioredoxin TRX Solubility-enhanced ubiquitous tags SNUT 17,000 Dalton protein SKP Monomeric bacteriophage T7 orc protein MOCR Escherichia coli secreted protein A ESPA N Utilization of substances NusA Protein G IgG domain B1 GB1 Protein A IgG repeat domain ZZ ZZ Mutated dehalogenase Halo Tags Bacteriophage T7 protein kinase T7PK Escherichia coli trypsin inhibitor Ecotin calcium-binding protein CaBP Stress response arsenate reductase ArsC N-terminal fragment of translation initiation factor IF2 IF2-domain 1 Stress response proteins RpoA Stress response proteins SlyD Stress response proteins Tsf Stress response proteins RpoS Stress response proteins PotD Stress response proteins Crr Escherichia coli acidic protein msyB Escherichia coli acidic protein yjD Escherichia coli acidic protein rpoD T7 phage tail P17 Metal binding proteins CUSF 53 amino acid long N-terminal extension sequence NEXT

[0139] Any fluorescent protein can be used. The term GFP is used herein to describe a group of green fluorescent proteins from different organisms, including the proteins sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP. The fluorescent protein can be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example, the peptide tag can be GFP 11 , the other peptide is GFP 1-10 The peptide tag can be a component of sfCherry. The peptide tag can be a component of sfCherry. 11 , the other peptide is sfCherry 1-10 In the presence of hydroxybenzylidene rhodanine analogs, peptide tags can be CFAST 11 or CFAST 10 , another peptide is CFAST. The peptide tag can be ccGFP 11 , the other peptide is ccGFP 1-10 .

[0140] Disclosed is a method for increasing the soluble yield of a protein of interest (POI) expressed in a cell-free system by expressing the protein of interest (POI) in the presence of a solubility-enhancing moiety, wherein the POI has a binding sequence that binds to the solubility-enhancing moiety, wherein the solubility-enhancing moiety comprises a component of a fluorescent protein and a moiety selected from those in the table above.

[0141] The improvement of protein solubility was tested to enhance the soluble expression yield of POI.

[0142] The fluorescent protein may be GFP. The fluorescent protein may be sfGFP. The fluorescent protein may be ccGFP. The solubility enhancing moiety may comprise ccGFP. 1-10 and MBP.

[0143] As the expressed protein binds to the binding partner, the protein can be assembled and thereby become fluorescent.Affinity interactions result in the two subcomponents of the fluorescent protein being close enough to each other to bind and induce fluorescence.

[0144] complementary GFP 11 The peptide amino acid sequence tag can be as follows:

[0145] 1. KRDHMVLLEFVTAAGITGT

[0146] 2. KRDHMVLHEFVTAAGITGT

[0147] 3. KRDHMVLHESVNAAGIT

[0148] 4. RDHMVLHEYVNAAGIT

[0149] 5. GDAVQIQEHAVAKYFTV

[0150] 6. GDTVQLQEHAVAKYFTV

[0151] 7. GETIQLQEHAVAKYFTE

[0152] 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 the fluorescence produced when the detection reagent is complementary to the expressed sequence. The protein will continue to fluoresce during the immobilization period, at which point the level of affinity purification can be determined.

[0153] Disclosed is a method for obtaining a GFP-containing 11 Label multiple droplets of protein and monitor GFP 11 Changes in tag levels are used to determine protein expression yield.

[0154] The method can measure aggregation over time by measuring solubility over time. For example,

[0155] 1. Mother droplets express POI.

[0156] 2. Split the mother droplet into multiple daughter droplets.

[0157] 3. At time 1, a droplet containing a detection agent is added to sub-droplet 1.

[0158] 4. At time 2, another droplet containing the detection agent is added to sub-droplet 2, where time 2 is greater than time 1.

[0159] 5. The complements from (3) and (4) each run for the same time period.

[0160] 6. Measure the fluorescence of the two droplets and compare them. The difference in fluorescence signal provides a measure of the apparent soluble protein yield.

[0161] Once a protein is expressed, expression can be halted by adding a chemical reagent to prevent further expression. Stability should be determined independently of further expression, as increasing the amount of protein with additional proteins can skew stability measurements. For example, the detection solution can be mixed with an agent that blocks protein synthesis, such as a Mg++ chelator (EDTA), to achieve integrated detection and blocking. Alternatively, the chelator / EDTA can be introduced first into all daughter droplets, and the detection agent can be introduced at different time points.

[0162] In addition to screening for optimal conditions for expression, the method can also be used to screen for optimal conditions for the stability of a particular protein. The target protein can be made with a variety of different amino acid additions as stabilizers. The expressed amino acids can be exposed to a variety of different times, temperatures, or environments, and the amount of remaining protein material can be determined. Therefore, in addition to screening for effective synthesis, effective stability conditions can also be identified. Once the optimal conditions are identified, they can be used for amplification and purification.

[0163] Screening and analysis can be performed in liquid reagent volumes, such as in microtiter plates or strip tubes. The reagent volume can be divided so that a portion is tested and a portion is reserved for future use.

[0164] 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 with a two-dimensional array of planar microelectrodes. The term excludes any device that simply has droplets in an oil flow in a channel. By activating specific electrodes, the droplets are moved across the surface by electrodynamic forces. Upon electrode activation, the dielectric layer becomes less hydrophobic, allowing the droplets to spread across 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.

[0165] The droplets may be aqueous droplets. The droplets may comprise an oil-immiscible organic solvent, such as, for example, DMSO. The droplets may be a mixture of water and a solvent, provided that the droplets do not dissolve in the bulk oil.

[0166] Digital microfluidics (DMF) refers to a two-dimensional planar platform for lab-on-a-chip systems 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. DMF can be used with analytical procedures such as mass spectrometry, colorimetry, electrochemistry, and electrochemiluminescence.

[0167] Any electrical motion method can be used to move droplets. Aqueous droplets can be moved using electrowetting on dielectrics (EWoD). EWoD is a variation of the electrowetting phenomenon in dielectric materials. During EWoD, a droplet of a conductive liquid is placed on an insulating, hydrophobic dielectric layer. Upon electrode activation, the dielectric layer becomes less hydrophobic, causing the droplet to spread across the surface.

[0168] The electrical signals on an EWoD or optically activated amorphous silicon (a-Si) EWoD device can be delivered via segmented electrodes, active-matrix thin-film transistors, or digital micromirrors. Optically activated s-Si EWoD devices are well known in the art and have been used to actuate droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0169] A source of supplemental oxygen can be supplied to the droplets. For example, during protein expression, droplets or bubbles containing gaseous or dissolved oxygen can merge with aqueous droplets. Alternatively, the source of oxygen can be a molecular source that releases oxygen. Alternatively, the droplets can be moved to the air / liquid boundary to increase diffusion of oxygen from the gaseous environment. Alternatively, the oil can be oxygenated.

[0170] Droplets can be formed before entering a microfluidic device and flow into the device. Alternatively, the droplets can merge on the device. A method is provided that includes merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free system having components for protein expression to form droplets.

[0171] Droplets can be moved on hydrophobic surfaces in digital microfluidics devices (ACS Nano 2018, 12, 6, 6050-6058). Hydrophobic surfaces can be materials such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGCChemicals Inc), or FluoroPel (Cytonix LLC). Hydrophobic surfaces can be modified to reduce biofouling, particularly due to exposure to CFPS reagents or nucleic acid reagents. Hydrophobic surfaces can also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Compared to typical fluorocarbon-based hydrophobic surfaces, superhydrophobic surfaces prevent biofouling. Thus, superhydrophobic surfaces extend the ability of digital microfluidics devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633-49648). The hydrophobic surface can also be a slippery liquid-infused porous surface (SLIPS), which can be formed by infusing a porous PTFE membrane with Krtox-103 oil (DuPont) (Lab Chip, 2019, 19, 2275).

[0172] In electrowetting on dielectrics (EWoD), the change in the contact angle of the reagent following application of a potential is an inverse function of surface tension. Therefore, for low-voltage EWoD runs, surface tension reduction is achieved by adding a surfactant to the reagents—which, in the case of CFPS reactions, means adding it to the lysate and to the DNA. This results in dilution of the lysate, which has been shown experimentally to lead to decreased expression levels of the target protein. Therefore, performing CFPS on DMF, in which a surfactant is added to the solution being moved, will inevitably result in dilution of the lysate and, therefore, decreased protein expression levels. Besides being a problem in itself, this further complicates extrapolating DMF results to in-vitro protein yield predictions. Another disadvantage of having to add a surfactant to the sample is that it increases the time required for sample preparation and increases the likelihood of inconsistent results due to 'user error' because there is more reagent handling. A further disadvantage of having to add a surfactant to the sample is that certain downstream operations are hampered. For example, if the target protein is being mixed with GFP in a cell-free system, 11 (or similar) peptide tag, which is expressed together with GFP 1-10 Downstream complementation of the detection polypeptide is hampered in the presence of surfactant.

[0173] Instead of adding a surfactant to the aqueous sample, a surfactant such as Span 85 (sorbitan trioleate) can be added to the oil. This has the advantage of allowing the CFPS reaction to be performed in DMF without dilution or doping. In addition, it simplifies the sample preparation process for setting up the reaction, improving ease of use and consistency of results. Using 1% weight / weight Span 85 in dodecane allows for undiluted CFPS reactions in DMF and undiluted detection of expressed non-fluorescent proteins. Surfactants other than Span 85 and oils other than dodecane can be used. A range of concentrations of Span 85 can be used. Surfactants can be nonionic, anionic, cationic, or amphoteric. Oils can be mineral or synthetic, including silicone, petroleum, and perfluorinated oils. Surfactants can have adverse effects on (1) the efficiency of the CFPS reaction and (2) the efficiency of the detection system (if the detection system involves complementation of the label and the detector). For example, by performing the CFPS reaction in DMF with an oil-surfactant mixture, detection of the expressed protein can also be performed without dilution and without the addition of aqueous surfactant. Surfactants have been shown to reduce the efficiency of some detection systems, including but not limited to the split GFP system, so it may be beneficial to remove the surfactant from the reagent mixture and add it to the oil instead.

[0174] Disclosed is a method for obtaining a GFP-containing 11 Label multiple droplets of protein and monitor GFP 11 A method for determining protein stability using changes in labeling levels. A method is disclosed comprising expressing a protein in a droplet on a digital microfluidics device having a two-dimensional array of planar microelectrodes, wherein the protein has GFP 11 peptide amino acid sequence tags, keeping the droplets at a fixed temperature, and monitoring the GFP in the droplets 11 The level of the amount of peptide amino acid sequence tags varies.

[0175] Device

[0176] Manipulation of droplets by applying an electric potential can be achieved on electrodes covered with an insulator or dielectric or an array of insulators or dielectrics. Manipulation of droplets by an applied electric potential is called electrowetting. Electrowetting occurs due to an inhomogeneous electric field that affects the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of a liquid on a solid surface (electrowetting on dielectric 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.

[0177] The electrowetting phenomenon occurs when a droplet is driven between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in surface tension, which causes the droplet to move due to a change in the contact angle of the droplet. The electrowetting effect can be quantitatively treated using the Young-Lippmann equation:

[0178] cosθ - cosθ0 = (1 / 2γLG) cV 2

[0179] where θ0 is the contact angle when the electric field across the interfacial layer is zero, γLG is the liquid-gas tension, and c is the specific capacitance (as ε r . ε0 / t is given, where ε r is the dielectric constant of the insulator / dielectric, ε0 is the permittivity of a vacuum, and t is the thickness. V is the applied voltage or potential. Therefore, the change in contact angle (inducing droplet movement) is a function of surface tension, potential, dielectric thickness, and dielectric constant.

[0180] When a droplet is driven by an EWoD, two opposing forces act on it: the electrowetting force induced by the electric field and the drag force, which consists of the drag force caused by the interaction of the droplet with the filler medium and the contact line friction (reference). The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all the drag forces is determined by the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε). r ) 1 / 2 Therefore, in order to reduce the driving voltage, it is necessary to reduce (t / ε r ) 1 / 2 (i.e., increasing the dielectric constant or reducing the thickness of the insulator / dielectric). To achieve low-voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high-quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is achieved, which is large enough to actuate the droplet. Therefore, most academic studies report the use of much higher voltages, >100V, to achieve electrowetting on easily fabricated thick dielectric films (>3 µm).

[0181] However, high-voltage EWoD-based devices with thick dielectric films have limited industrial applicability, primarily due to their limited droplet multiplexing capabilities. The use of low-voltage devices, including thin-film transistors (TFTs) and optically activated amorphous silicon (a-Si) layers, has paved the way for industrial applications of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplexed manner. However, the driving voltages of TFTs or optically activated a-Si are low (typically <15 V). A bottleneck in the fabrication and, therefore, adoption of low-voltage devices has been the technical challenge of depositing high-quality thin-film insulators / dielectrics. Consequently, there is a significant need to improve the fabrication and composition of thin-film insulator / dielectric devices.

[0182] Typically, electrodes (or array elements) used in EWoDs are coated with either (i) a hydrophilic insulator / dielectric and a hydrophobic coating, or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings include fluoropolymers such as Teflon AF 1600 or CYTOP. These materials, as hydrophobic coatings on dielectrics, are typically less than 100 nm thick and may contain defects in the form of pinholes or porous structures; therefore, it is crucial that the insulator / dielectric is pinhole-free to avoid electrical shorts. Teflon has also been used as an insulator / dielectric, but due to its low dielectric constant and the thickness required to be pinhole-free, it has higher voltage requirements. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on silicones, epoxies (e.g., SU-8), or parylenes (e.g., parylene N, parylene C, parylene D, or parylene HT). Teflon is also used as a hydrophobic overcoat for these insulator / dielectric polymers due to its minimal contact angle hysteresis and high contact angle with aqueous solutions. However, reliably fabricating pinhole-free coatings of parylene or SU-8 <1 micron presents difficulties; therefore, the thickness of these materials is generally maintained at 2-5 microns, at the expense of increased electrowetting voltage requirements. Conventional EWoD devices with parylene C have also been reported to be susceptible to breakage and instability for repeated droplet manipulation using cell culture media. Multilayer insulator devices deposited from metal oxide and parylene C films have been used to create more robust insulators / dielectrics and capable of operating 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 insulators / dielectrics for EWoD devices. They offer the advantage of thin-film deposition (<100 nm) using standard cleanroom methods. However, these materials are inherently hydrophilic, requiring additional hydrophobic coatings, and are susceptible to pinhole formation due to the thin-film layer deposition process. Together with the need for lower voltage operation of EWoDs, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using insulators / dielectrics with high dielectric constants) and (2) optimizing fabrication methods to make the insulators / dielectrics pinhole-free to avoid dielectric breakdown.

[0183] The operation of EWoD devices is affected by contact angle saturation and hysteresis, which is believed to be caused by any one or a combination of the following phenomena: (1) charge entrapment in the hydrophobic membrane or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instability, (4) dielectric breakdown of the dielectric layer, (5) electrode-electrode-insulator interface capacitance (due to double layer effects), and (6) surface fouling (such as by biomolecules). One of the adverse effects of such hysteresis is a reduced operational lifetime of EWoD-based devices.

[0184] Contact angle hysteresis is believed to be the result of charge accumulation at the interface or within the hydrophobic insulator after a few runs. This charging phenomenon increases the required driving voltage, leading to eventual catastrophic dielectric breakdown. The most likely explanation is that pinholes in the insulator / dielectric may allow the liquid to come into contact with the electrode, leading to electrolysis. A hydrophobic insulator that is prone to pinholes or porous further promotes electrolysis.

[0185] Most studies used to understand contact angle hysteresis on EWoDs have been based on short timescales and conducted with low conductivity solutions. Long duration actuation (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCl) may produce several effects other than electrolysis. Ions in the solution can permeate through the hydrophobic coating (under an applied electric field) and interact with the underlying insulator / dielectric. Ion penetration can lead to (1) changes in the dielectric constant due to charge trapping (which is different from interfacial charging) and (2) changes in the surface potential of the pH-sensitive metal oxide. Both can lead to a reduction in the electrowetting force for manipulating aqueous droplets, resulting in contact angle hysteresis. The present inventors have previously found that damage from high conductivity solutions reduces electrowetting on electrodes by inhibiting the modulation of the contact angle under applied electric field, or making electrowetting impossible on electrodes.

[0186] An electrokinetic device includes a first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating, the functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate 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.

[0187] The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may have a thickness of 10 nm to 100 μm. A combination of more than one material may be used, and the dielectric layer may include more than one sublayer, each of which may be made of different materials.

[0188] The conformal layer can comprise parylene, silicone, or epoxy. It can be a thin, protective parylene coating between an insulating dielectric and a hydrophobic coating. Parylene is typically 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 rather to serve as a conformal layer between the dielectric and hydrophobic layers. The inventors have discovered that, unlike other similar insulating coatings of the same thickness, such as PDMS (polydimethylsiloxane), parylene prevents contact angle hysteresis caused by highly conductive solutions or solutions that deviate from neutral pH for extended periods. The thickness of the conformal layer can range from 10 nm to 100 μm. The thickness of the conformal layer can range from 100 nm to 300 nm.

[0189] The hydrophobic layer may comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite, a zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, a carbon nanotube structure, a silica nanocoating, or a slippery liquid-infused porous coating.

[0190] The element may include one or more of a plurality of array elements, each element containing element circuitry; discrete electrodes; a thin film semiconductor whose electrical properties are modulated by incident light; and a thin film photoconductor whose properties are modulated by incident light.

[0191] The functional coating may comprise 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.

[0192] The electrokinetic device may include a controller for regulating the voltage supplied to individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each thin film transistor is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows each individual element to be individually controlled.

[0193] The second substrate may further include a second hydrophobic layer disposed on the second electrode. The first substrate and the second substrate may be disposed such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electromotion working space between the hydrophobic layers.

[0194] The method is particularly suitable for aqueous droplets with a volume of 1 μL or less.

[0195] The EWoD-based devices shown and described below are active-matrix thin-film transistor devices containing a thin-film dielectric coating with a Teflon hydrophobic surface coating. These devices are based on devices described in E Ink's patent application entitled "Digital microfluidic device including dual substrates with thin-film transistors and capacitive sensing," filed as U.S. Patent Application No. 2019 / 0111433, which is incorporated herein by reference.

[0196] An electrokinetic device is described herein, comprising:

[0197] A first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating comprising:

[0198] a dielectric layer in contact with the matrix electrodes,

[0199] a conformal layer in contact with the dielectric layer, and

[0200] a hydrophobic layer in contact with the conformal layer;

[0201] a second substrate including a top electrode;

[0202] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0203] A voltage source is operatively coupled to the matrix electrodes.

[0204] This document describes an electric motion device comprising:

[0205] A first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating comprising:

[0206] one or more dielectric layers comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes,

[0207] a conformal layer comprising parylene in contact with the dielectric layer, and

[0208] a hydrophobic layer in contact with the conformal layer;

[0209] a second substrate including a top electrode;

[0210] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0211] A voltage source is operatively coupled to the matrix electrodes.

[0212] The electrokinetic device may be used with other components, such as, for example, means for heating and cooling the device or a kit for introducing reagents as needed.

[0213] A "droplet" refers to a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by a carrier fluid and / or, in some cases, a gas or gas mixture, such as ambient air. For example, a droplet can be completely surrounded by a carrier fluid, or can be bounded by a carrier fluid and one or more surfaces of an EWoD device. Droplets can take a variety of shapes; non-limiting examples generally include disks, slugs, truncated spheres, ellipsoids, spheres, partially compressed spheres, hemispheres, ovoids, cylinders, and various shapes formed during droplet manipulation, such as merging or splitting, or as a result of such shapes coming into contact 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 both aqueous and non-aqueous components. Droplets can also comprise dispersions and suspensions, such as magnetic beads in an aqueous solvent. In various embodiments, the droplets may 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, stool samples, fluids containing single or multiple cells, fluids containing organelles, fluidized tissue, fluidized organisms, fluids containing multicellular organisms, biological swabs, and biological washes.

[0214] "Droplet operations" refers to any operation on one or more droplets on a microfluidic device. Droplet operations may include, for example: loading a droplet into a DMF device; dispensing one or more droplets from a source reservoir; splitting, separating, or splitting 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 operations described herein; and / or any combination of the foregoing. The terms "merge," "merging," "combine," "combining," and the like are used to describe the creation of a single droplet from two or more droplets. It should be understood that when such terms are used with respect to two or more droplets, any combination of droplet operations sufficient to result in the combination of the two or more droplets into a single droplet may be used. For example, "merging droplet A with droplet B" can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting 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 volume of the resulting droplets (i.e., the volumes of the resulting droplets can be the same or different) or the number of resulting droplets (i.e., the number of resulting droplets can be 2, 3, 4, 5, or more). The term "mixing" refers to a droplet operation that results in a more uniform distribution of one or more components within a droplet. Examples of "loading" droplet operations include, but are not limited to, microdialysis loading, pressure-assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations can be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and / or hydrophobic regions on a surface and / or by physical barriers.

[0215] Experiments and results

[0216] Figure 5 Shown are experimental results for 24 different proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstituted cell-free protein synthesis system. Each construct contained GFP 11 In the row labeled "Filter," GFP 1-10 The test substance is present from the beginning of expression. The row marked "end point" shows the absence of GFP. 1-10 The detection substance was expressed for 10 hours and then GFP was used 1-10Fluorescence signal was measured after 5 hours of complementation of the substances. This experiment showed significant differences between expression / complementation in the "Screening Bio-Ink" compared to the "end-point" assay. Protein clusters formed after expression were detected only using the endpoint assay, indicating that the protein aggregated after expression, thereby reducing the soluble yield. Aggregates were clearly visible in several constructs, indicating that protein aggregates are likely present. The level of aggregation can identify conditions worth further testing and identify conditions with high levels of aggregation, from which further purification of material is unlikely.

[0217] Figure 6-8 Shown are the expression results of two proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstituted cell-free protein synthesis system. Eight different cell-free expression blends were tested for each protein. Figure 6 White light images after 10 hours of expression are shown. Figure 7 White light images after addition of the test protein (another 5 h) are shown. Figure 8 Fluorescence (blue light) images after adding the test protein are shown. For the soluble protein (DHFR) (left side of the array), the droplets appear uniform in all three images because the expressed protein does not appear in the form of clumps, and no aggregation or aggregation is observed in any of the three images. In the central three columns of the images after expression ( Figure 6 ), protein clusters (P17_IFN) can be detected in the optical image. After adding the detection protein, clusters can still be detected in the white light image ( Figure 7 ). Fluorescence image ( Figure 8 ) shows fluorescent clusters within the droplets, indicating the presence of protein aggregates. Droplets with high levels of turbidity in the white light image also have high levels of signal from bound detection protein, demonstrating that the protein can be detected using either white light or fluorescence measurements.

Claims

1. A method for determining the degree of aggregation of proteins in one or more droplets on a digital microfluidics device, wherein the degree of aggregation is measured using optical methods.

2. The method of claim 1 for determining the degree of aggregation of a protein expressed in a cell-free system, comprising expressing a protein of interest (POI) in one or more droplets on a digital microfluidics device, wherein the degree of aggregation of the expressed POI is measured using an optical method.

3. The method of claim 1 or claim 2, wherein the ratio of soluble POI to insoluble POI is determined.

4. The method according to any one of claims 1 to 3, wherein the degree of aggregation is measured by counting the number of aggregates, the area of ​​aggregates, the intensity of aggregates, or by measuring turbidity or dispersion.

5. The method of any one of claims 1 to 4, wherein the POI produces aggregates detectable by light scattering. The method of claim 5 , wherein the light is measured at an angle different from that of incident light.

7. A method according to any one of claims 1 to 6, comprising adding further droplets containing a substance that binds to the POI to enhance the signal from the POI.

8. The method according to any one of claims 1 to 7, wherein the POI has a binding sequence that binds to a detection moiety.

9. A method according to claim 8, comprising adding a further droplet containing a detection moiety to the droplet containing the POI to produce a detectable signal in the combined droplet.

10. The method of claim 8 or 9, wherein the binding sequence contains four or more amino acids.

11. The method of any one of claims 7 to 10, wherein the detection moiety is a protein.

12. The method of claim 11, wherein the detection moiety comprises a component of a fluorescent protein.

13. The method of claim 12, wherein the detection moiety comprises a component of a fluorescent protein and an additional solubility enhancer.

14. The method of claim 13, wherein the solubility enhancer comprises a moiety selected from the group consisting of: 。 15. The method of claim 14, wherein the detection moiety comprises ccGFP 1-10 and MBP.

16. The method of claim 10, wherein the binding sequence is a component of a fluorescent protein.

17. A method according to any preceding claim, wherein the protein of interest has ccGFP 11 Label.

18. A method according to any preceding claim, wherein the concentration of the detection moiety exceeds the POI.

19. The method of any one of claims 2 to 18, wherein the expression is performed for at least 3 hours before the binding moiety or substance that binds to the aggregate is added to the droplets.

20. A method according to any preceding claim, wherein the POI comprises a ccGFP selected from the group consisting of 11 Peptide amino acid sequence tag: KRDHMVLLEFVTAAGITGT KRDHMVLHEFVTAAGITGT KRDHMVLHESVNAAGIT RDHMVLHEYVNAAGIT GDAVQIQEHAVAKYFTV GDTVQLQEHAVAKYFTV GETIQLQEHAVAKYFTE.

21. The method according to any one of claims 2 to 20, wherein the expression is performed using cell-free lysate.

22. The method of any one of claims 2 to 20, wherein the expression is performed using assembled components for transcription and translation in the Purified Recombinant Elements (PURE) system.

23. The method according to any one of claims 1 to 22, wherein the digital microfluidics device comprises an oil-filled or humidified gas environment, wherein the humidified gas environment is achieved by closing or sealing the digital microfluidics device and providing an onboard reagent reservoir.

24. A method according to any preceding claim comprising 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 ccGFP 11 Peptide amino acid sequence tag containing ccGFP is added after the expression process 1-10 The droplets were sampled and the uniformity of the fluorescent signal was measured to determine the degree of aggregation of the expressed protein.

25. A method according to any preceding claim, comprising monitoring the extent of aggregation under various conditions for stability screening at different chemical compositions or temperatures.

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