WO2025253020A2 - Dosages sur des protéines exprimées - Google Patents

Dosages sur des protéines exprimées

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Publication number
WO2025253020A2
WO2025253020A2 PCT/EP2025/065975 EP2025065975W WO2025253020A2 WO 2025253020 A2 WO2025253020 A2 WO 2025253020A2 EP 2025065975 W EP2025065975 W EP 2025065975W WO 2025253020 A2 WO2025253020 A2 WO 2025253020A2
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WIPO (PCT)
Prior art keywords
tag
protein
droplets
proteins
substrate
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Pending
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PCT/EP2025/065975
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WO2025253020A3 (fr
Inventor
Michael Chun Hao CHEN
Florian MITTELBERGER
Tobias William Barr Ost
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Nuclera Ltd
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Nuclera Ltd
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Publication of WO2025253020A2 publication Critical patent/WO2025253020A2/fr
Publication of WO2025253020A3 publication Critical patent/WO2025253020A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

  • compositions for the on-device expression and activity assays on the expressed proteins are provided herein.
  • the methods are applicable to performing protein assays on a microfluidic device.
  • the invention relates to methods for expression and assays of proteins on a microfluidic device in cellular or cell-free systems.
  • Cell-free protein synthesis (CFPS) regimes are alternatives to cellbased expression systems and can be treated as reagents rather than organisms, making them amenable to in-vitro experimentation techniques. Additionally, cell-free systems are less sensitive to toxic protein synthesis; are open systems that can be modulated via addition of elements due to the lack of a cell membrane; are adaptable to high-throughput experiments; and can be used to good effect in small volumes.
  • CFPS Cell-free protein synthesis
  • Efficient protein synthesis relies on having the correct nucleic acid expression construct in the correct conditions. Protein synthesis and purification can be improved by attaching additional amino acids to the protein of interest, for example sequences improving solubility or tags for purification. In order to efficiently screen the optimal cell-free conditions for expression of a particular protein sequences it is desirable to provide a population of nucleic acid expression constructs. Furthermore, in order to identify the best DNA construct to generate a protein of interest it is desirable to provide a population of nucleic acid expression constructs. The invention herein describes methods for the screening of nucleic acid constructs suitable for cell- free protein expression, and the use thereof.
  • Proteins of interest may be expressed as a fusion to a fluorescent protein, such as green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • MBP maltose-binding protein
  • WO2022/038353 describes a method for measuring protein expression levels using split fluorescent protein systems.
  • the level of expressed protein is measured in the presence of an excess of detector species, thereby measuring a single interaction which assembles a fluorescent protein.
  • W02024003538 relates to assays where proteins are measured for binding affinity. Expressed proteins can be bound to binding species, and upon binding generate a signal.
  • Suitable assays may include cleavage of a substrate having a fluorophore-quencher pair, for example a peptide or nucleic acid sequence which may be cleaved by a protease or nuclease.
  • Suitable assays may include extension assays where a substrate is acted on by a ligase or polymerase with subsequent detection of the elongated substrate. Described herein are such assays performed on a digital microfluidic device.
  • Disclosed is a method for assaying the activity of a synthesised protein on a digital microfluidic device comprising: i) synthesising a protein on a digital microfluidic device, and ii) performing an activity assay using the synthesised protein wherein the activity assay generates a detectable signal on the digital microfluidic device.
  • the signal is only generated if the protein is correctly expressed and is correctly folded and active.
  • the expression may be achieved in cells.
  • the expression may use cell-free protein synthesis (CFPS). After expression, the synthesised protein may be purified on the digital microfluidic device.
  • CFPS cell-free protein synthesis
  • the expressed protein has activity against a substrate which can be measured upon expression.
  • the expressed protein may be a protease, nuclease, polymerase or ligase.
  • Detection assays may involve a detectable signal generated by separation of a fluorophore and quencher pair.
  • the fluorophore and quencher pair may be attached via a nucleotide or amino acid sequence.
  • the synthesised protein may have nuclease or protease activity in order to cleave the substrate and separate the fluorophore and quencher.
  • Detection assays may involve extension of a substrate, for example a nucleic acid primer.
  • the synthesised protein may be a nucleic acid polymerase or ligase and the detectable signal may be generated by extension of a priming sequence.
  • the extension may be performed using monomers such as nucleoside triphosphates or via ligation of further nucleic acid strands.
  • Detection assays may involve circularisation of a substrate using a ligase.
  • Assays may be performed on the crude material after protein synthesis or the proteins may be purified from the expression components.
  • the proteins may be expressed on the device and captured onto solid supports, for example magnetic beads. Magnetic beads may also be used to immobilise the substrates before or after the activity assay.
  • the activity assay may be performed in a single composition such that the synthesised protein cleaves a substrate that is present in the mixture as the protein is synthesised.
  • the substrate may be added after expression once the protein is synthesised and optionally purified.
  • the detectable signal may be an increase or decrease in fluorescence.
  • Suitable assays may include assays where the detectable signal is generated by capturing of an extended substrate on a solid support.
  • Suitable assays may include assays where the detectable signal is generated by cleaving of a substrate on a solid support to release a signal moiety into solution.
  • Any expression system can be used to express the proteins.
  • the transcription and translation may use a mammalian, insect, plant or bacterial derived expression system.
  • the expression may be achieved in a cellular or cell-free system.
  • the cell-free system may be a lysate or reconstituted system. Suitable systems may include a human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary (CHO) lysate system, a wheat germ cell-free system, a E. coli whole cell lysate system, a tobacco cell lysate system, a yeast lysate system or in a system of purified recombinant elements (PURE) or a mixture thereof.
  • RRL rabbit reticulocyte lysate
  • CHO Chinese Hamster Ovary
  • Suitable cellular systems may include a human system, a rabbit reticulocyte system, a Chinese Hamster Ovary (CHO) system, a wheat germ system, a E. coli whole cell system, a tobacco system or a yeast system. Where a cellular system is used, the cells may be lysed on device to release the synthesised protein prior to the activity assay. The reagents may be blended or supplemented to optimise expression.
  • Disclosed herein is a method comprising the steps of: a. expressing target proteins in droplets on a digital microfluidic device having an array of electrodes; b. binding the expressed proteins to magnetic beads; c. immobilising and washing the beads to remove unbound proteins; d. optionally eluting the immobilised proteins from the beads; e. adding a substrate to the purified proteins; and f. measuring a detectable signal caused by the purified protein acting on the substrate.
  • a method comprising the steps of: a. expressing target proteins in droplets on a digital microfluidic device having an array of electrodes, wherein the droplets contain a substrate on which the synthesised proteins can act; and b. measuring a detectable signal caused by the synthesised protein acting on the substrate.
  • kits comprising: i) a digital microfluidic device, ii) a nucleic acid sequence for expressing a protease, nuclease, polymerase or ligase, iii) reagents for expressing the nucleic acid sequence, and iv) a substrate comprising a fluorophore and a quencher pair measuring the activity of the expressed protease, nuclease, polymerase or ligase.
  • the kit may further comprising a solid support such as magnetic beads for binding to the expressed sequence.
  • the solid support may be magnetic or paramagnetic beads.
  • the beads may contain agents which bind to the proteins allowing capture and purification.
  • the synthesised proteins may carry a detectable tag such as a binding agent or a sub-component of a fluorescent protein.
  • the detectable tag may contain for example amino acid sequences from GFPn, sfGFPu or ccGFPn.
  • the detector species may bind to the tag in order to produce a signal or to capture the label.
  • the detector species may be for example GFPi-io, sfGFPi-io or ccGFPi-io.
  • Assays may be performed in parallel on multiple species.
  • a population of (n) expressed proteins may be expressed in separate droplets, a population of (m) potential substrates supplied in separate droplets and the droplets of expressed proteins split into at least (m) number of droplets and substrates split into at least (n) number droplets and the droplets combined to perform (n)x(m) number of binding assays simultaneously on the device.
  • the protein may be captured onto beads using a binding interaction.
  • the proteins may have a purification tag selected from:
  • Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)
  • the fluorescent protein may be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano.
  • the peptide tag may be GFPn and the further polypeptide GFPi-io.
  • the peptide tag may be one component of sfCherry.
  • the peptide tag may be sfCherryn and the further polypeptide sfCherryi-w.
  • the peptide tag may be CFASTn or CFASTio and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog.
  • the peptide tag may be ccGFPn and the further polypeptide ccGFPi-io.
  • the peptide tag may be sfGFPn and the further polypeptide sfGFPi-io.
  • the fluorescent protein may be GFP.
  • the fluorescent protein may be sfGFP.
  • the fluorescent protein may be ccGFP.
  • the complementary ccGFP/GFPn peptide amino acid sequence could be the following:
  • Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.
  • GFPn or GFPi-io can be fused to the protein of interest or binding partner through an amino acid linker.
  • the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.
  • the assays may be performed in a digital microfluidic device, for example an electrowetting-on- dielectric (EWoD) device.
  • the expression may be performed in droplets on the digital microfluidic device having an array of electrodes.
  • the device may be for example an array of active-matrix thin film transistors (AM-TFTs).
  • the droplets may be on the device in an oil layer, which may contain a surfactant.
  • the droplets are typically aqueous droplets in a hydrophobic oil layer.
  • One or both of the layers may contain a surfactant.
  • the surfactant in the oil layer may be a non-ionic surfactant.
  • the surfactant in the oil layer may be a sorbitan ester such as Span85.
  • the expressed protein may be fused to multiple tags to aid purification, solubility or detection.
  • the protein may be fused to multiple GFPn peptide tags and the synthesis occurs in the presence of multiple GFPi-io polypeptides.
  • the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi. 10 polypeptides.
  • the protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more sfCherryno polypeptides and one or more GFPi-io polypeptides.
  • Figure 1 shows an activity assay.
  • a quenched substrate can be cleaved using an expressed protease, thus generating a signal.
  • Figure 2 shows the concentration profile for the assay of Figure 1. Concentrations of TEV protease higher than 50 pM can be clearly seen.
  • Figure 3 shows TEV production and activity on device. The activity was measured as fluorescence. The error bars indicate the standard deviation. The concentration of NEB TEV was loaded at 200 nM for the final droplet concentration to be 50 nM.
  • Figure 4 shows BFL and FFL images from run. The columns are labelled with the constructs produced in the columns.
  • a method for determining protein activity using a fluorescent readout in a droplet on a digital microfluidic device having an array of electrodes comprising: i) synthesising a protein on a digital microfluidic device, and ii) performing an activity assay using the synthesised protein wherein the activity assay generates a detectable signal on the digital microfluidic device.
  • activity assays may include for example:
  • Colorimetric Assays which measure the change in colour resulting from protease activity.
  • Commonly used colorimetric substrates include:
  • Azocasein When hydrolyzed by proteases, it releases dye, which can be measured at specific wavelengths (e.g., 440 nm).
  • p-Nitroaniline (pNA) Derivatives These substrates release pNA upon hydrolysis, producing a yellow colour that can be quantified by measuring absorbance at 405 nm.
  • Fluorometric Assays which are highly sensitive and measure the increase in fluorescence resulting from protease activity.
  • Fluorogenic substrates commonly used include 4- Methylumbelliferyl (MUF) Derivatives: These release a fluorescent product (MUF) upon hydrolysis, which can be measured at specific wavelengths (excitation at 360 nm and emission at 450 nm).
  • Fluorescence Resonance Energy Transfer (FRET) assays utilize substrates labelled with a donor and acceptor fluorophore (or quencher). Protease activity separates the fluorophore and quencher, leading to a change in fluorescence that can be measured.
  • FRET Fluorescence Resonance Energy Transfer
  • Turbidimetric Assays which measure the change in turbidity resulting from protease-mediated hydrolysis of insoluble substrates like casein or haemoglobin. The reduction in turbidity is quantified by measuring light scattering or absorbance at specific wavelengths.
  • activity assays may include for example:
  • Fluorometric Assays which measure the change in fluorescence resulting from nuclease activity on fluorogenic substrates.
  • Fluorescently Labelled Nucleic Acids DNA or RNA substrates labelled with fluorophores such as FAM (fluorescein) or TMR (tetramethylrhodamine). Cleavage results in an increase in fluorescence that can be measured.
  • SYBR Green and SYBR Gold Assay SYBR Green and SYBR Gold bind to double-stranded DNA, and fluorescence decreases upon DNA degradation.
  • Fluorescence Resonance Energy Transfer (FRET) assays use substrates labelled with a donor and acceptor fluorophore or quencher. Cleavage of the substrate changes the FRET efficiency, altering the fluorescence signal.
  • Donor and acceptor fluorophores are typically placed at opposite ends of dual-labelled oligonucleotides. Nuclease activity separates the fluorophores, leading to an increase in donor fluorescence and a decrease in FRET.
  • the shortened strands may be captured for example by hybridisation.
  • the nucleic acid substrate may be immobilised, either before or after cleavage in order to release or capture the detectable label.
  • the nuclease may be an exonuclease or endonuclease and may be active on double stranded or single stranded templates. Where double stranded substrates are cleaved, the labels may be on opposing strands such that the signal is increased once the shorted strands denature.
  • the nuclease may be a RNA-guided DNA endonuclease enzyme such as a CAS nuclease.
  • a guide RNA may be used to direct cleavage of a nucleic acid substrate.
  • the device may be used to express and measure activity of CAS nucleases such as engineered CAS9 nuclease enzymes.
  • activity assays may include for example:
  • assays measure the increase in fluorescence resulting from ligase activity on fluorogenic substrates.
  • substrates labelled with fluorophores such as FAM (fluorescein) or TMR (tetramethylrhodamine). Ligation results in an increase in fluorescence that can be measured.
  • molecular beacons Hairpin- shaped oligonucleotides with a fluorophore and quencher. Upon ligation, the hairpin structure is disrupted, increasing fluorescence.
  • Fluorescence Resonance Energy Transfer (FRET) assays use substrates labelled with a donor and acceptor fluorophore. Ligation of the substrate changes the FRET efficiency, altering the fluorescence signal. For example using dual-labelled oligonucleotides: Donor and acceptor fluorophores are placed at opposite ends. Ligation brings the fluorophores closer, leading to changes in FRET signal.
  • FRET Fluorescence Resonance Energy Transfer
  • chromophores that are sensitive to the length of the template such as SYBR green or SYBR Gold can be used to determine activity.
  • the substrate can be joined to form a circular construct in the presence of the ligase
  • the presence of the circular construct can be detected, thereby indicating ligase activity.
  • the extended strands may be captured for example by hybridisation.
  • the nucleic acid substrate may be immobilised, either before or after cleavage in order to release or capture the detectable label.
  • activity assays may include for example:
  • Fluorometric Assays which measure the increase in fluorescence resulting from polymerase activity.
  • fluorescently labelled nucleotides Incorporation of labelled nucleotides (e.g., FAM, TMR) into the growing DNA or RNA strand increases fluorescence or a SYBR Green or SYBR Gold Assay: SYBR Green binds to double-stranded DNA, and fluorescence increases as the polymerase synthesizes new DNA.
  • SYBR Gold is an asymmetrical cyanine dye which can be used as a stain for double-stranded DNA, single-stranded DNA, and RNA.
  • the SYBR Gold assay may be use to determine terminal transferase activity by adding nucleotides to an initiator substrate in a template independent fashion.
  • the extended initiator gives an increased signal compared to the starting initiator.
  • the level of the expressed protein may be determined using a fluorescence based assay, for example using a split fluorescent protein. Any fluorescent protein may be used.
  • the fluorescent protein may be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano.
  • the peptide tag may be GFPn and the further polypeptide GFPi-io.
  • the peptide tag may be one component of sfCherry.
  • the peptide tag may be sfCherryn and the further polypeptide sfCherryi-io.
  • the peptide tag may be CFASTn or CFASTio and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog.
  • the peptide tag may be ccGFPn and the further polypeptide ccGFPi-io.
  • the fluorescent protein may be GFP.
  • the fluorescent protein may be sfGFP.
  • the fluorescent protein may be ccGFP.
  • CFPS cell-free protein synthesis
  • the synthesised protein can be any protein of interest.
  • the protein can be a protease, nuclease, ligase or polymerase.
  • the assay can be performed on a microfluidic devices using electrowetting droplet operations.
  • Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field.
  • Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting and optoelectrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing.
  • Electrodes covered with a dielectric layer each of which are connected to an A/C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path.
  • DMF digital microfluidics
  • DMF utilizes alternating currents on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation.
  • optoelectrowetting which uses light to photo-actuate the surface to change the hydrophobicity.
  • Cell-free protein synthesis also known as in-vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells.
  • the in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in-vitro transcription.
  • CFPS has been known for decades, and many commercial systems are available.
  • Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol.
  • EWoD electrowetting-on-dielectric
  • electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other bioproducts adsorb and/or denature to hydrophobic surfaces.
  • Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Langmuir 2011, 27 , 13, 8586-8594).
  • Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air.
  • the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops.
  • air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature suitable for enzyme activity and the duration of the synthesis needs to be prolonged for synthesized proteins levels to be detectable. Protein expression typically requires an ample supply of oxygen.
  • the components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.
  • the droplet can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed.
  • the droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed.
  • the droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed.
  • the act of moving the droplet allows oxygen to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.
  • the droplet can be moved using any means of electrowetting.
  • the droplet can be moved using electrowetting-on-dielectric (EWoD).
  • EWoD electrowetting-on-dielectric
  • the electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.
  • the oil in the device can be any water immiscible liquid.
  • the oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil.
  • DMPS dodecamethylpentasiloxane
  • the oil can be oxygenated prior to or during the expression process.
  • the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.
  • humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device.
  • the aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C).
  • a source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the droplets during the protein expression. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coli cell growth (RSCAdv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.
  • the droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.
  • the droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with the binding partner droplets for screening.
  • the cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression.
  • Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes.
  • a cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc.
  • the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.
  • nucleic acid template can be expressed using the system described herein.
  • Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA.
  • Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS.
  • mRNA can be produced through in-vitro transcription systems.
  • the methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.
  • An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process.
  • the cell-free extract having the components for protein expression includes everything required for protein expression apart from the nucleic acid template. Thus the term includes all the relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, protein expression is initiated without further reagents being required.
  • the cell-lysate can be supplemented with additional reagents prior to the template being added.
  • the cell-free extract having the components for protein expression would typically be produced as a bulk reagent or 'master mix' which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets.
  • Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available.
  • EAE E. coli
  • RRL rabbit reticulocytes
  • WGE wheat germ
  • insect cells ICE
  • Yeast Kluyveromyces the D2P system
  • the cell-free system can be assembled from the required reagents.
  • Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied.
  • the PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes.
  • the protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.
  • digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes.
  • the term excludes any devices simply having droplets in a flow of oil in a channel.
  • the droplets are moved over the surface by electrokinetic forces by activation of particular electrodes.
  • the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.
  • a digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage.
  • additional reagents can be supplied by merging the original droplet with a second droplet.
  • the second droplet can carry any desired additional reagents, including for example oxygen or 'power' sources, or test reagents to which it is desired to expose to the expressed protein.
  • the droplets can be aqueous droplets.
  • the droplets can contain an oil immiscible organic solvent such as for example DMSO.
  • the droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil.
  • the droplets can be in a bulk oil layer.
  • a dry gaseous environment simply dries the bubbles onto the surface during the expression process, leaving comet type smears of dried material by evaporation.
  • the device is filled with liquid for the expression process.
  • the aqueous droplets can be in a humidified gaseous environment.
  • a device filled with air can be sealed and humidified in order to provide an environment that reduces evaporation of CFPS droplets.
  • the droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets.
  • the templates can be added by merging droplets on the microfluidic device.
  • the templates can be added to the droplets outside the device and then flowed into the device for the expression process.
  • the expression process can be initiated on the device by increasing the temperature.
  • the expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.
  • the expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours.
  • the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet.
  • the moving can be continuous, or can be repeated with intervening periods of nonmovement.
  • the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed.
  • the aqueous droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed.
  • the aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed.
  • the act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet.
  • the act of moving improves the level of protein expression over a droplet which remains static.
  • Digital microfluidics refers to a two-dimensional planar surface platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.
  • the droplet can be moved using any means of electrowetting.
  • the aqueous droplet can be moved using electrowetting-on-dielectric (EWoD).
  • Electrowetting on a dielectric is a variant of the electrowetting phenomenon that is based on dielectric materials.
  • EWoD Electrowetting on a dielectric
  • a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.
  • the electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors.
  • Optically-activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).
  • the oil in the device can be any water immiscible or hydrophobic liquid.
  • the oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil.
  • DMPS dodecamethylpentasiloxane
  • the air in the device can be any humidified gas.
  • a source of supplemental oxygen can be supplied to the droplets.
  • droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression.
  • the source of oxygen can be a molecular source which releases oxygen.
  • the droplets can be moved to an air/liquid boundary to enable increased diffusion of oxygen from a gaseous environment.
  • the oil can be oxygenated.
  • the droplets can be presented in a humidified air filled device.
  • an affinity tag such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag
  • CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin.
  • renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS.
  • CF continuous flow
  • CE continuous exchange
  • Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces.
  • droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011, 27, 13, 8586-8594).
  • Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and/or Pluronic F127.
  • droplets containing CFPS components may contain TWEEN 20 at 0.1% v/v, Triton X-100 at 0.1% v/v, and/or Pluronic F127 at 0.05% w/v.
  • An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to 'user error,' as there is more handling of reagents.
  • An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFPn (or similar) peptide tag, it's downstream complementation with a GFPi-io (or similar) detector polypeptide is hindered in the presence of surfactant. Removal of the surfactant from the aqueous phase is therefore advantageous.
  • surfactant such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil.
  • Span85 e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025
  • This has the advantages of enabling CFPS reactions to proceed on-DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results.
  • Using 1% w/w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins.
  • surfactants besides Span85, and oils other than dodecane could be used.
  • a range of concentrations of Span85 could be used.
  • Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof.
  • Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils.
  • Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on-DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant.
  • Affinity tags may be appended to proteins so that they can be purified from their crude biological source using an affinity technique.
  • the purification tags may be selected from for example FLAG- tag, His-tag, GST-tag, MBP-tag, STREP-tag.
  • the Flag® tag also known as the DYKDDDDK-tag, is a popular protein tag that is commonly used in affinity chromatography and protein research. His tags are polyhistidine strings of amino acids, typically between 6 and 9 histidine amino acids in length.
  • the immobilisation or purification tag can be attached to the C or N terminus of the protein.
  • the protein may be fused to multiple tags.
  • the protein can be produced with a tag which enables immobilisation.
  • the protein may be expressed to have an amino acid binding sequences.
  • Affinity/immobilisation tag may be selected from:
  • Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)
  • VSV-tag (YTDIEMNRLGK)
  • the proteins may be immobilised directly onto the surface of the device.
  • the proteins may be immobilised onto beads or particles, for example magnetic or paramagnetic beads.
  • the beads may contain the binding partner for the chosen tag, for example a metal ion to chelate His or streptavidin or strep-tactin to bind to the strep-tags.
  • the material may be bound to the beads on the device, of suitable particles already bound to proteins may be added to the device and moved to discreet locations.
  • the substrate may be for example a dual labelled peptide or nucleic acid sequence having a fluorophore and quencher system which can be separated by cleavage activity of the expressed protein.
  • the substrate may be one of more nucleic acid sequences which can be ligated together or circularised.
  • the substrate may be a nucleic acid sequence which can be extended by polymerase activity, either in templated or untemplated form. Polymerase activity may incorporate labelled or unlabelled nucleotides. Incorporation of labelled nucleotides for example may allow capture of a labelled substrate. For example the attachment of biotin dNTP's onto a fluorescent substrate may allow capture of the resulting extended material. Capture of the fluorescently labelled material is an indication of protein activity.
  • assays can be performed. Examples of the types of assays that can be implemented include cleavage to release unquenched fluorescence or elongation to generate a detectable substrate. Assays may be performed by either capturing the generated signal, for example by hybridisation, or via cleavage of an immobilised substrate to generate signal in solution.
  • Electrowetting occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD).
  • DEP can also be used to create forces on polarizable particles to induce their movement.
  • the electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal.
  • EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric.
  • the electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle.
  • an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction (ref).
  • the minimum voltage applied to balance the electrowetting force with the sum of all drag forces is variably determined by the thickness-to-dielectric contact ratio of the insulator/dielectric, (t/£ r ) 1/2 .
  • it is required to reduce (t/£ r ) 1/2 (i.e., increase dielectric constant or decrease insulator/dielectric thickness).
  • thin insulator/dielectric layers must be used.
  • the deposition of high quality thin insulator/dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved.
  • Most academic studies thus report the use of much higher voltages >100V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.
  • High voltage EWoD-based devices with thick dielectric films have limited industrial applicability largely due to their limited droplet multiplexing capability.
  • the use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a- Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion.
  • the driving voltage for TFTs or optically-activated a-Si are low (typically ⁇ 15 V).
  • the bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators/dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator/dielectric devices.
  • the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator/dielectric and a hydrophobic coating or (ii) a hydrophobic insulator/dielectric.
  • a hydrophilic insulator/dielectric and a hydrophobic coating or (ii) a hydrophobic insulator/dielectric.
  • Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP.
  • the thickness of this material as a hydrophobic coating on the dielectric is typically ⁇ 100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator/dielectric is pinhole free to avoid electrical shorting.
  • Teflon has also been used as an insulator/dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free.
  • Other hydrophobic insulator/dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. SU-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator/dielectric polymers.
  • EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator/dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules).
  • contact angle saturation and hysteresis which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator/dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules).
  • An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes.
  • the dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate.
  • the dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.
  • the conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation/dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours.
  • the conformal layer may be between 10 nm and 100 pm thick.
  • the conformal layer may be between 100 nm and 200 nm thick.
  • the hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.
  • the elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.
  • the functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer.
  • the electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes.
  • the electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled.
  • the second substrate may also comprise a second hydrophobic layer disposed on the second electrode.
  • the first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.
  • the method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.
  • EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on "Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing", US patent application no 2019/0111433, incorporated herein by reference.
  • electrokinetic devices including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;
  • an electrokinetic device including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;
  • electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.
  • Droplet refers to a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by carrier fluid and/or, in some instances, a gas or gaseous mixture such as ambient air.
  • a droplet may be completely surrounded by carrier fluid or may be bounded by carrier fluid and one or more surfaces of an EWoD device.
  • Droplets may take a wide variety of shapes; non-limiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more working surface of an EWoD device.
  • Droplets may include typical polar fluids such as water, as is the case for aqueous or non-aqueous compositions, or may be mixtures or emulsions including aqueous and non-aqueous components. Droplets may also include dispersions and suspensions, for example magnetic beads in an aqueous solvent.
  • a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes.
  • a biological sample such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, ex
  • Droplet operation refers to any manipulation of one or more droplets on a microfluidic device.
  • a droplet operation may, for example, include: loading a droplet into the DMF device; dispensing one or more droplets from a source reservoir; splitting, separating or dividing a droplet into two or more droplets; moving a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a microfluidic device; other droplet operations described herein; and/or any combination of the foregoing.
  • merge “merge,” “merging,” “combine,” “combining” and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, “merging droplet A with droplet B,” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other.
  • splitting is not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e., the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more).
  • mixing refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading” droplet operations includes but is not limited to microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles.
  • TEV Protease can be measured on device.
  • TEV protease can break down a quenched peptide substrate sequence ENLYFQG (AnaSpec, Sensolyte 520 TEV Protease Assay kit, AS-72227) allowing the fluorescent signal from the FAM unit, which is quenched when the peptide is intact.
  • ENLYFQG AnaSpec, Sensolyte 520 TEV Protease Assay kit, AS-722257
  • the experiment is run on a Nuclera EProtein DiscoveryTM system with blue light imaging at 30 °C.
  • a script is used whereby 49 px 2 droplets from A10 are mixed with droplets from A12 of the same size, BIO + B12 etc. to generate 8X different conditions.
  • the aim was to show that TEV protease produced on a Nuclera eProtein DiscoveryTM device was active.
  • the aim was to load DNA encoding TEV protease, mix with CFB, and detect activity using a substrate that when cleaved by TEV protease has a fluorescent signal.
  • Nucleic acid templates (eGenesTM) were loaded into ports Al to A8 and Bl to B8.
  • the eGenes had the standard solubility tags.
  • Ports Cl to C4 were loaded with buffer blank.
  • Ports C5 to C8 were loaded with commercial TEV protease at 50 nM (NEB TEV).
  • TEV protease from NEB was supplied at a concentration of 35710 nM. This was diluted 1 in 10 in lx eGene buffer to 3571 nM. 0.5 pl of this dilution was mixed with 79.5 pl of lx eGene buffer.
  • the expression ports were loaded with a cell-free expression mix with no additive, TRXB1, DnaK or GSSG.
  • the wash, elution, and bead ports were loaded with wash buffer.
  • the controls were loaded as standard.
  • the expression mixture plus egenes were incubated for protein production, then droplets of a TEV substrate (5-FAM/QXL520) in 4x TEV protease buffer were added.
  • the 4x TEV protease buffer was added to ensure the DTT was present to create a reducing environment required for TEV function.
  • the fluorescence was analysed and the intensity was plotted (Fig. 3).
  • TEV protease was produced and active on device. There was active TEV protease with droplets that were turbid or had clusters present. The presence of DnaK or GSSG in the expression mixture improved TEV activity, this was for both TEV produced on device and externally sourced TEV.

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Abstract

L'invention concerne des procédés et des compositions pour l'analyse de l'activité de protéines. Les procédés sont applicables à la synthèse de protéines sur un dispositif microfluidique et à des dosages à l'aide des protéines exprimées.
PCT/EP2025/065975 2024-06-07 2025-06-09 Dosages sur des protéines exprimées Pending WO2025253020A2 (fr)

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Citations (4)

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