METHOD OF ELECTROWETTING

Abstract

A method for moving an aqueous droplet comprising providing 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: 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 confornial 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 niatrix electrodes. The method further comprises disposing an aqueous droplet on a first matrix electrode; and providing a differential electrical potential between the first matrix electrode and a second matrix electrode with the voltage source, thereby moving the aqueous droplet.

Claims

1-22. (canceled)

23. An electrokinetic device for moving an aqueous droplet, comprising: a first substrate having a matrix of electrodes, wherein each of the matrix of electrodes is coupled to a thin film transistor, and wherein the matrix of electrodes are overcoated with a functional coating comprising: a dielectric in contact with the matrix of electrodes comprising hafnium oxide; a conformal layer comprising parylene in contact with the dielectric; a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode and a second hydrophobic layer; 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 of electrodes, the voltage source controllable to provide a differential electrical potential between a first matrix electrode and a second matrix electrode in order to move the aqueous droplet between the first matrix electrode and the second matrix electrode.

24. The electrokinetic device of claim 23, wherein the dielectric comprises multiple layers.

25. The electrokinetic device of claim 23, the dielectric has a thickness between 10 nm and 100 μm.

26. The electrokinetic device of claim 23, wherein the conformal layer has a thickness between 10 nm and 100 μm.

27. The electrokinetic device of claim 23, wherein the hydrophobic layer comprises 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.

28. The electrokinetic device of claim 23, wherein the dielectric of the functional coating includes a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer.

29. The electrokinetic device of claim 23, further comprising a controller to control the differential electrical potential provided between the first matrix electrode and the second matrix electrode.

30. The electrokinetic device of claim 23, further comprising a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to one of the plurality of scan lines, and one of the plurality of gate lines, and the plurality of gate lines are operatively connected to the controller.

31. The electrokinetic device of claim 23, wherein the aqueous droplet has a volume of 1 μL or smaller.

32. The electrokinetic device of claim 23, wherein the voltage source being further controllable to provide the differential electrical potential between a third matrix electrode and the second matrix electrode thereby causing the aqueous droplet to contact a second aqueous droplet on the third matrix electrode.

33. The electrokinetic device of claim 23, wherein the dielectric comprises one or more sublayers of different materials.

34. The electrokinetic device of claim 33, wherein the dielectric comprises three sublayers.

35. The electrokinetic device of claim 33, wherein the dielectric comprises: a first layer including an aluminum oxide or a hafnium oxide, the first layer having a thickness between 9 nm and 80 nm; a second layer including a tantalum oxide or a hafnium oxide, the second layer having a thickness between 40 nm and 250 nm; and a third layer including a tantalum oxide or a hafnium oxide, the third layer having a thickness between 5 nm and 60 nm, wherein the second layer is disposed between the first layer and the third layer.

36. The electrokinetic device of claim 23, wherein the hydrophobic layer comprises an amorphous fluoropolymer.

Description

FIGURES

[0052] FIG. 1 shows cross sectional schematic for a traditional EWoD device;

[0053] FIG. 2 shows a cross section of a device according to the invention;

[0054] FIG. 3 depicts a device according to the invention with voltages applied and droplets;

[0055] FIG. 4 depicts an active matrix as used in conjunction with the invention;

[0056] FIG. 5A shows degradation of array elements on a device without any conformal layer;

[0057] FIG. 5B shows an array of elements coated in parylene C and without any defects; and

[0058] FIG. 6 depicts an image sequence demonstrating droplet formation on a device according to the invention.

DETAILED DESCRIPTION

[0059] FIG. 1 depicts a conventional electrowetting device with a substrate 10 and a plurality of individually controllable elements 11. The individually controllable elements may be arranged in an array such that multiple droplets may be manipulated simultaneously. The electrical properties of the individually controllable elements 11 can be varied. For example, each individually controllable element may comprise an electrode or a circuit. As shown in FIG. 1, each individually controllable element is connected to a voltage source. Alternatively, each element may comprise a thin film semiconductor in which the electrical properties can be modulated by incident light or a thin film photoconductor whose properties can be modulated by incident light.

[0060] Covering the individually controllable elements 11 is a dielectric layer 12. As an alternative to the dielectric layer 12 there may be an insulator. The insulator/dielectric may be made of SiO.sub.2, silicon oxynitride, Si.sub.3N.sub.4, 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, barium strontium titanate, parylene siloxane, epoxy or a mixture thereof. The insulator/dielectric layer has a thickness of 10-10,000 nm.

[0061] On top of the insulator 12 (or dielectric) is a hydrophobic coat 13. The hydrophobic coat may comprise a fluoropolymer such as, for example, Teflon, CYTOP or PTFE. The hydrophobic coating layer may be made of an amorphous fluoropolymer or siloxane or organic silane. The hydrophobic layer has a thickness of 1-1,000 nm.

[0062] A second electrode 14 is positioned opposite the array of individually controllable elements and the second electrode and the individually controllable elements are separated by a spacer which defines an electrokinetic workspace.

[0063] FIG. 2 depicts an electrowetting device according to the invention in which, on top of the individually controllable elements is a functional coating comprising three component pans: a dielectric layer 12, a conformal layer 30 and a hydrophobic layer 13. According to an embodiment the conformal coat is made of parylene, or preferably parylene C. The conformal layer 30 has a thickness of 10-10,000 nm and prevents ions from interacting with the insulator/dielectric layer 12. The second electrode 14 may comprise a second hydrophobic layer facing the (first) hydrophobic layer. The electrokinetic workspace is then formed between the hydrophobic layers.

[0064] In order to promote adhesion between the different layer gaseous precursors are often used. This can be used when the layers are deposited using a spin coating or a dip coating.

[0065] An aqueous solution of 1M is applied to the substrate and a voltage applied. Through the application of a voltage the aqueous solution forms droplets 35 above the individually controllable elements, as shown in FIG. 3.

[0066] FIG. 4 depicts an array of individually controllable elements forming an electrode array 202. FIG. 4 is a diagrammatic view of an exemplary driving system 200 for controlling droplet operation by an AM-EWoD propulsion electrode array 202. The AM-EWoD driving system 200 may be in the form of an integrated circuit adhered to a support plate. The elements of the EWoD device are arranged in the form of a matrix having a plurality of data lines and a plurality of gate lines. Each element of the matrix contains a TFT for controlling the electrode potential of a corresponding electrode, and each TFT is connected to one of the gate lines and one of the data lines. The electrode of the element is indicated as a capacitor Cp. The storage capacitor Cs is arranged in parallel with Cp and is not separately shown in FIG. 4.

[0067] The controller shown comprises a microcontroller 204 including control logic and switching logic. It receives input data relating to droplet operations to be performed from the input data lines 22. The microcontroller has an output for each data line of the EWoD matrix, providing a data signal. A data signal line 206 connects each output to a data line of the matrix. The microcontroller also has an output for each gate line of the matrix, providing a gate line selection signal. A gate signal line 208 connects each output to a gate line of the matrix. A data line driver 210 and a gate line driver 212 is arranged in each data and gate signal line, respectively. The figure shows the signals lines only for those data lines and gate lines shown in the figure. The gate line drivers may be integrated in a single integrated circuit. Similarly, the data line drivers may be integrated in a single integrated circuit. The integrated circuit may include the complete gate driver assembly together with the microcontroller.

[0068] The integrated circuit may be integrated on a support plate of the AM-EWoD device. The integrated circuit may include the entire AM-EWoD device driving system.

[0069] The data line drivers provide the signal levels corresponding to a droplet operation. The gate line drivers provide the signals for selecting the gate line of which the electrodes are to be actuated. A sequence of voltages of one of the data line drivers 210 is shown in FIG. 4

[0070] As illustrated in FIG. 4, traditional AM-EWoD cells use line-at-a-time addressing, in which one gate line n is high while all the others are low. The signals on all of the data lines are then transferred to all of the pixels in row n. At the end of the line time gate line n signal goes low and the next gate line n+1 goes high, so that data for the next line is transferred to the TFT pixels in row n+1. This continues with all of the gate lines being scanned sequentially so the whole matrix is driven. This is the same method that is used in almost all AM-LCDs, such as mobile phone screens, laptop screens and LC-TVs, whereby TFTs control the voltage maintained across the liquid crystal layer, and in AM-EPDs (electrophoretic displays).

[0071] FIG. 5A depicts an array of elements on an AM-EWoD device without a conformal layer. A driving voltage has been applied to high ionic strength solutions and, as can be seen, results in damage and defects around the edge of some of the elements. An example is highlighted in a dotted line box. The result of this damage is failure of to perform EWoD actuation of an aqueous droplet in the area, further failure of an aqueous droplet to wet the area, and/or also general failures to dispense or split from an existing droplet to form two droplets.

[0072] FIG. 5B shows an array of elements, similar to those depicted in FIG. 5A but coated in parylene C. Again, a driving voltage has been applied to high ionic strength droplets but did not result in the defects seen in FIG. 5A. The result of the conformal coating is the lack of damage seen in FIG. 5A resulting in the ability of an aqueous droplet to wet the area and/or dispense or split from an existing droplet to form two droplets in areas of an AM-EWoD device contacted by high ionic strength droplets.

[0073] Experimental Details

[0074] Adhesion Promotion

[0075] Adding 0.5% v/v Silane A-174 to a 1:1 ratio of isopropanol/water and stirring for 30 seconds formed solution 1. Solution 1 was left to stand for at least 2 hours to fully react and was used within 24 hours. Substrates were immersed in the Solution 1 for 30 minutes, while ensuring the flex strips of the TFT arrays were kept dry. Substrates were removed and air dried for 15 minutes and then cleaned in isopropanol for 15-30 seconds with agitation using tweezers. Substrates were dried with an air gun and stored in a Teflon box for Parylene C coating within 30 hours.

[0076] Parylene Coating

[0077] Prepared substrates (silanised and non-silanised) were arranged face up on a rotating stage alongside a clean glass slide within the deposition chamber of a thoroughly clean SCS Labcoter 2 and the chamber was sealed. 50 mg of Parylene C dimer was weighed into a disposable aluminium boat and loaded into the sublimation chamber. The system was sealed and pumped down to 50 milliTorr before liquid nitrogen was added to the cold trap. The system continued to evacuate throughout the deposition process. The sublimation chamber was heated to 175° C. and the heater cycled to maintain a target pressure of 0.1 Torr. The sublimation chamber was connected to the deposition chamber by a pyrolysis zone which was heated to 690° C. at a target pressure of 0.5 Torr. The deposition zone remained at ambient temperature, circa 25° C., and around 50 milliTorr. The system was maintained at temperature and pressure for two hours. The system was allowed to return gradually to ambient temperature over 30-40 minutes before the stage and vacuum pump were turned off and the system vented. The samples were removed from the deposition chamber and the coating thickness verified as circa 100 nm by profilometry.

[0078] The device was then subjected to 22 hours of continuous operation with a high salt solution. FIG. 6 depicts the reliable dispensation of a droplet through electrowetting actuation even after 22 hours of continuous operation (dispensing electrowetting actuation shown from FIG. 6 top left to top middle to top right images), as opposed to an AM-EWoD device shown in FIG. 5A. Even after this the droplet can be moved over the continuously actuated area (shown in FIG. 6 bottom left to bottom middle to bottom right images).

[0079] Applications of the Invention

[0080] The invention can be used in a myriad of different applications. In particular the invention can be used to move cells, nucleic acids, nucleic acid templates, proteins, initiation oligonucleotide sequences for nucleic acid synthesis, beads, magnetic beads, cells immobilised on magnetic beads, or biopolymers immobilised on magnetic beads.

[0081] In these applications the steps of disposing an aqueous droplet having an ionic strength on a first matrix electrode and providing a differential electrical potential may be repeated many times. They may be repeated over 1000 times or over 10,000 times, sometimes over a 24 hour period.

[0082] The present method can be used in the synthesis of nucleic acids, such as phosphoramidite-based nucleic acid synthesis, templated or non-templated enzymatic nucleic acid synthesis, or more specifically, terminal deoxynucleotidyl transferase (TdT) mediated addition of 3′-O-reversibly terminated nucleoside 5′-triphosphates to the 3′-end of 5′-immobilized nucleic acids. During enzymatic nucleic acid synthesis, the following steps are taken on the instrument: [0083] I. Addition solution containing TdT, optionally pyrophosphatase (PPiase), 3′-O-reversibly terminated dNTPs, and required buffer (including salts and necessary reaction components such as metal divalents) is brought to a reaction zone containing an immobilized nucleic acid, where the nucleic acid is immobilized on a surface such as through magnetic beads via a covalent linkage to the 5′ terminus of the nucleic acid. The initial immobilized nucleic acid may be known as an initiator oligonucleotides and comprises N nucleotides, for example 3-100 nucleotides, preferably 10-80 nucleotides, and more preferably 20-65 nucleotides. Initiator oligonucleotides may contain a cleavage site, such as a restriction site or a non-canonical DNA base such as U or 8-oxoG. Addition solution may optionally contain a phosphate sensor, such as E. coli phosphate-binding protein conjugated to MDCC fluorophore, to assess the quality of nucleic acid synthesis as a fluorescent output. dNTPs can be combined in ratios to make DNA libraries, such as NNK syntheses. [0084] II. Wash solution, either in bulk or in discrete droplets, is applied to reaction zones to wash away the addition solution. Wash solution typically has a high solute concentration (>1 M NaCl). [0085] III. Deprotection solution, either in bulk or in discrete droplets, is applied to reaction zones to deprotect the 3′-O-reversible terminator added to the immobilized nucleic acids in the immobilized nucleic acid zone in step I. Deprotection solution typically has a high solute concentration. [0086] IV. Wash solution, either in bulk or in discrete droplets, is applied to reaction zones to wash away the deprotection solution. [0087] V. Steps I-IV are repeated until desired sequences are synthesized, for example steps I-IV are repeated 10, 50, 100, 200 or 1000 times.

[0088] The present method can be used in the preparation of oligonucleotide sequences, either via synthesis or assembly. The device allows synthesis and movement of defined sequences. Using the present method the initiation sequences can be modified at a specific location above an electrode and the extended oligonucleotides prepared. The initiation sequences at different locations can be exposed to different nucleotides, thereby synthesising different sequences in different regions of the electrokinetic device.

[0089] After synthesis of a defined population of different sequences in different regions of the electrokinetic device, the sequences can be further assembled in longer contiguous sequences by joining two or more synthesised strands together.

[0090] Described herein is a method for preparing a contiguous oligonucleotide sequence of at least 2n bases in length comprising taking the electrokinetic device as described herein having a plurality of immobilised initiation oligonucleotide sequences, one or more of which contains a cleavage site, using the initiation oligonucleotide sequences to synthesise a plurality of immobilised oligonucleotide sequences of at least n bases in length, using cycles of extension of reversibly blocked nucleotide monomers, selectively cleaving at least two of the immobilised oligonucleotide sequences of least n bases in length into a reaction solution whilst leaving one or more of the immobilised oligonucleotide sequences attached, hybridizing at least two of the cleaved oligonucleotides to each other, to form a splint, and hybridizing one end of the splint to one of the immobilized oligonucleotide sequences and joining at least one of the cleaved oligonucleotides to the immobilised oligonucleotide sequences, thereby preparing a contiguous oligonucleotide sequence of at least 2n bases in length.

[0091] The steps of synthesis and assembly may involve high solute concentrations where the ionic strength would degrade the devices without the protecting conformal layer.

[0092] The method of moving aqueous droplets may also be used to help facilitate cell-free expression of peptides or proteins. In particular, droplets containing a nucleic acid template and a cell-free system having components for protein expression in an oil-filled environment can be moved using a method of the invention in the described electrokinetic device.

[0093] The present invention can be used to automate the movements of droplets in a cartridge. For example, droplets intended for analysis can be moved according to the present invention. The present invention could be incorporated into a cartridge used for local clinician diagnostics. For example it could be used in conjunction with nucleic acid amplification testing (NAAT) to determine nucleic acid targets in, for example, genetic testing for indications such as cancer biomarkers, pathogen testing for example detecting bacteria in a blood sample or virus detection, such as a coronavirus, e.g. SARS-CoV-2 for the diagnosis of COVID-19.

[0094] The device may be thermocycled to enable nucleic acid amplification, or the device may be held at a desired temperature for isothermal amplification. Having different sequences synthesised in different regions of the device allows multiplex amplification using different primers in different regions of the device.

[0095] Furthermore the invention can be used in conjunction with next generation sequencing in which DNA is synthesised by the addition of nucleotides and large numbers of samples are sequenced in parallel. The present invention can be used to accurately locate the individual samples used in next generation sequencing.

[0096] The invention can be used to automate library preparation for next generation sequencing. For example the steps of ligation of sequencing adaptors can be carried out on the device. Amplification of a selective subset of sequences from a sample can then have adaptors attached to enable sequencing of the amplified population.

[0097] Where used herein “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0098] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0099] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.