Electro-wetting method

Abstract

A method for moving an aqueous droplet, comprising providing an electrokinetic device comprising 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 coated 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 and second substrates and defining an electrokinetic working space; and a voltage source operably coupled to the matrix electrodes. The method further comprises disposing an aqueous droplet on the first matrix electrode; and providing a differential potential between the first and second matrix electrodes with the voltage source, thereby moving the aqueous droplet.

Description

[0001] Field of the invention

[0002] The present invention is in the field of fluid electrodynamics: electrowetting on dielectrics (EWoD) and dielectrophoresis (DEP); and devices using these phenomena. The present invention relates to enhancing the performance and durability of operation and lifetime of devices by coating a conformal layer on top of a dielectric or insulator stack.

[0003] BACKGROUND

[0004] Manipulation of small droplets can be achieved on electrodes covered with an insulator or dielectric or a series of insulators or dielectrics by applying an electric potential. The manipulation of droplets as a result of the applied electric potential is known as electrowetting. The electrodynamic origin is either the non-uniform electric field affecting the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or the change in the contact angle of a liquid on a solid surface (electrowetting on dielectrics or EWoD). DEP can also be used to generate forces on polarizable particles to induce their motion. The electrical signal can be transmitted to discrete electrodes, transistors, transistor arrays or semiconductor films whose electrical properties can be modulated by optical signals. The EWoD phenomenon occurs when a small droplet is actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion due to a change in the droplet contact angle. The electrowetting effect can be quantitatively treated using the Young-Lippmann equation:

[0005]

[0006] where θ0is the contact angle at zero electric field on the interface layer, γLGis the liquid-gas tension, c is the specific capacitance (given by ε r / t, where ε r is the dielectric constant of the insulator / dielectric, ε0is the dielectric constant of vacuum, and t is the thickness), and V is the applied voltage or potential. Thus, the change in the contact angle (inducing droplet movement) is a function of the surface tension, the potential, the dielectric thickness, and the dielectric constant.

[0007] When a droplet is actuated by EWoD, there are two opposing sets of forces acting on it: the electrowetting force induced by the electric field and the opposing force including the drag force and the contact line friction force (referenced) generated by the droplet interaction with the filler medium. The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all drag forces is variably determined by the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε r ) 1 / 2 Thus, to reduce the actuation voltage, it is necessary to reduce (t / ε r ) 1 / 2(i.e., increasing the dielectric constant or decreasing the insulator / dielectric thickness). To enable low voltage driving, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before reaching the desired electro wetting contact angle that is large enough to drive the droplets. Therefore, most academic studies report the use of much higher voltages > 100 V on thick dielectric films (> 3 pm) that are easy to fabricate to enable electro wetting.

[0008] However, 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 (TFTs) and photo-activated amorphous silicon layers (a-Si) pave the way for industrial applications of EWoD based devices due to their greater flexibility in addressing electrical signals in a highly multiplexed manner. The driving voltage of TFTs or photo-activated a-Si is low (typically < 15 V). The bottleneck in fabricating and thus employing low voltage devices has been the technical challenge of depositing high quality thin film insulators / dielectrics. Therefore, there has been a particular need to improve the fabrication and composition of thin film insulator / dielectric devices.

[0009] Generally, the electrodes (or array elements) used in EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating, or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings include fluoropolymer such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on a dielectric is typically < 100 nm, and can have defects in the form of pinholes or porous structures; thus, it is particularly important that the insulator / dielectric be pinhole-free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but 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). Teflon is still used as a hydrophobic topcoat for these insulator / dielectric polymers due to minimal contact angle hysteresis and higher contact angles with aqueous solutions. However, there are difficulties in reliably making pinhole-free parylene or SU-8 coatings < 1 micron; thus, the thickness of these materials is generally kept at 2-5 microns at the cost of increased electrowetting voltage requirements. It has also been reported that traditional EWoD devices using parylene C are prone to damage and unstable for repeated droplet manipulation using cell culture media. Multilayer insulator devices with deposited metal oxide and parylene C films have been used to make more robust insulator / dielectrics and enable operation at lower applied voltages. Inorganic materials such as metal oxides and semiconductor oxides, commonly used as "gate dielectrics" in the CMOS industry, have been used as insulator / dielectrics for EWoD devices. They offer the advantage of thin film deposition (< 100 nm) using standard cleanroom processes. These materials are inherently hydrophilic, require an additional hydrophobic coating, and are prone to pinhole formation due to the thin film layer deposition process. In conjunction with the need for lower voltage operation for EWoD, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using high dielectric constant insulator / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole-free to avoid dielectric breakdown.

[0010] Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be caused by one or a combination of the following phenomena: (1) trapping of electrical charges in the hydrophobic film 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 (caused by double layer effects), and (6) fouling of the surface (such as by biological macromolecules). One of the adverse effects of this hysteresis is a reduced operational lifetime of the EWoD-based device.

[0011] Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after multiple operations. Due to this charging phenomenon, the required driving voltage increases, leading to eventual catastrophic dielectric breakdown. The most likely explanation is that pinholes at the insulator / dielectric can bring the liquid into contact with the electrode, causing electrolysis. The ease of forming pinholes or porous hydrophobic insulators further promotes electrolysis.

[0012] Most studies to understand contact angle hysteresis on EWoD have been conducted at short timescales and with low conductivity solutions. Long time driving (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCl) can produce several effects in addition to electrolysis. Ions in the solution (under the applied electric field) can penetrate through the hydrophobic coating and interact with the underlying insulator / dielectric. Ion penetration can lead to (1) a change in the dielectric constant due to charge trapping (which is different from interface charging) and (2) a change in the surface potential of pH-sensitive metal oxides. Both can lead to a reduction in the electrowetting forces used to manipulate aqueous droplets, resulting in contact angle hysteresis. The inventors have found that the damage from high conductivity solutions reduces or disables electrowetting on the electrodes by suppressing the modulation of the contact angle when an electric field is applied.

[0013] It is therefore an object of the present invention to provide a method for preventing contact angle saturation and hysteresis. SUMMARY

[0015] According to the present invention, there is provided a method for moving an aqueous droplet, comprising providing an electrokinetic device comprising 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 coated 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 and second substrates and defining an electrokinetic working space; and a voltage source operably coupled to the matrix electrodes. The method further comprises disposing an aqueous droplet on a first matrix electrode; and providing a differential potential between the first and second matrix electrodes with the voltage source, thereby moving the aqueous droplet.

[0016] The inventors have found that contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH can be mitigated by depositing a conformal layer. This method and device can be used at ionic strengths greater than 0.1 M and greater than 1.0 M.

[0017] The inventors have found that contact angle hysteresis on EWoD-based devices caused by high conductivity solutions or solutions deviating from neutral pH can be mitigated by depositing a thin protective parylene coating between the insulating dielectric and the hydrophobic coating.

[0018] The ability to continuously drive high ionic strength solutions for long periods of time provides great utility to those wishing to perform certain biochemical processes and experiments. High ionic strength solutions are commonly used as wash buffers to disrupt interactions of nucleic acids and proteins, for example in commonly performed chromatin immunoprecipitation (ChIP) assays. High ionic strength solutions can also be used for osmotic cell lysis. Additionally, cultivation of seaweed is generally performed in media that is isotonic with seawater having an ionic strength of 600-700 mM. Another application of high ionic strength solutions is for elution of proteins from affinity matrices after purification. High ionic strength buffers are also used for enzymatic nucleic acid synthesis. A variety of high ionic strength solutions (1000 mM monovalent or higher) can be used during both wash and deprotection steps in the process of enzymatic DNA synthesis.

[0019] The dielectric layer can comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer can have a thickness of 10 nm to 100 pm. Combinations of more than one material can be used, and the dielectric layer can include more than one sublayer which can be different materials.

[0020] Exemplary layers can be found in application WO2020226985. The dielectric layer of the present invention can be deposited on a substrate, for example a substrate comprising a plurality of electrodes disposed between the substrate and the layered dielectric. In some embodiments, the electrodes are disposed in an array and each electrode is associated with a thin film transistor (TFT). In some embodiments, a hydrophobic layer is deposited on the third layer, i.e. on top of the dielectric stack. In some embodiments, the hydrophobic layer is a fluorine-containing polymer, which can be 10 to 50 nm thick, and is deposited using spin coating or another coating method. Also described herein is a method for producing a layered dielectric of the type described above. The method comprises providing a substrate, depositing a first layer using atomic layer deposition (ALD), depositing a second layer using sputtering, and depositing a third layer using ALD. (The first layer is deposited on the substrate, the second layer is deposited on the first layer, and the third layer is deposited on the second layer). The first ALD layer generally comprises aluminum oxide or hafnium oxide and has a thickness of 9 nm to 80 nm. The second sputtered layer can comprise tantalum oxide or hafnium oxide and has a thickness of 40 nm to 250 nm. The third ALD layer generally comprises tantalum oxide or hafnium oxide and has a thickness of 5 nm to 60 nm. In some embodiments, the atomic layer deposition comprises plasma-assisted atomic layer deposition. In some embodiments, the sputtering comprises radio frequency magnetron sputtering. In some embodiments, the method further comprises spin coating a hydrophobic material on the third layer.

[0021] Optionally, the dielectric 'layer' can comprise multiple layers. The first layer can comprise aluminium oxide or hafnium oxide and have a thickness of 9 nm to 80 nm. The second layer can comprise tantalum oxide or hafnium oxide and have a thickness of 40 nm to 250 nm. The third layer can comprise tantalum oxide or hafnium oxide and have a thickness of 5 nm to 60 nm. The second and third layers can comprise different materials, for example, the second layer can comprise primarily hafnium oxide while the third layer comprises primarily tantalum oxide. Alternatively, the second layer can comprise primarily tantalum oxide while the third layer comprises primarily hafnium oxide. In some embodiments, the first layer can be aluminium oxide. In preferred embodiments, the first layer is 20 to 40 nm thick, while the second layer is 100 to 150 nm thick and the third layer is 10 to 35 nm thick. The thickness of each layer can be measured using a variety of techniques including, but not limited to, scanning electron microscopy, ion beam backscattering, X-ray scattering, transmission electron microscopy and ellipsometry.

[0022] The conformal layer can comprise parylene, silicone or epoxy. It can be a thin protective parylene coating between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In the present invention, the rationale for depositing parylene is not to improve insulating / dielectric properties, such as reducing pinholes, but as a conformal layer between the dielectric layer and the hydrophobic layer. The inventors have found that parylene, in contrast to other similar insulating coatings such as PDMS (polydimethylsiloxane) of the same thickness, prevents contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for long periods. The thickness of the conformal layer can be 10 nm to 100 μιη.

[0023] A method for moving aqueous droplets is disclosed, the method comprising:

[0024] An electrokinetic device is provided, the electrokinetic device comprising:

[0025] a first substrate having a matrix of electrodes, 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:

[0026] one or more dielectric layers comprising silicon nitride, hafnium oxide or aluminium oxide in contact with the matrix electrodes,

[0027] a conformal layer comprising parylene in contact with the dielectric layers, and

[0028] a hydrophobic layer in contact with the conformal layer;

[0029] a second substrate comprising a top electrode;

[0030] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0031] a voltage source operably coupled to the matrix electrode;

[0032] providing an aqueous droplet on a first matrix electrode; and

[0033] providing a differential potential between the first matrix electrode and a second matrix electrode with the voltage source, thereby moving the aqueous droplet between the first matrix electrode and the second matrix electrode.

[0034] The hydrophobic layer can comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite, a zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structures, a silica nano-coating, or a smooth liquid-impregnated porous coating.

[0035] The elements can comprise one or more of a plurality of array elements, each element comprising an element circuit; a discrete electrode; a thin film semiconductor whose electrical properties are modulated by incident light; a thin film photoconductor whose properties are modulated by incident light.

[0036] The functional coating can 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.

[0037] The electrokinetic device can comprise a controller for regulating the voltage provided to individual matrix electrodes. The electrokinetic device can comprise 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 operably connected to the controller. This allows all individual elements to be controlled individually.

[0038] The second substrate can further comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates can be disposed such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electrokinetic working space between the hydrophobic layers.

[0039] The method is particularly suitable for aqueous droplets having a volume of 1 pL or less.

[0040] The invention can be used to bring adjacent aqueous droplets into contact by disposing a second aqueous droplet on a third matrix electrode and providing a differential potential between the third matrix electrode and the second matrix electrode with the voltage source.

[0041] The invention further provides an assay, nucleic acid synthesis, nucleic acid assembly, nucleic acid amplification, nucleic acid manipulation, next generation sequencing library preparation, protein synthesis, or cell manipulation comprising repeating the above method steps.

[0042] In particular, the steps of setting an aqueous droplet on the first matrix electrode and providing a differential potential are repeated multiple times. The movement of the droplet can be repeated more than 1000 times or more than 10000 times. These method steps can be repeated more than 1000 times within 24 hours.

[0043] The EWoD-based devices shown and described below are active matrix thin film transistor devices comprising a thin film dielectric coating and a Teflon hydrophobic top coating. These devices are based on the devices described in E Ink Corporation patent application “Digital microfluidic devices including dual substrates with thin film transistors and capacitive sensing” U.S. Patent Application No. 2019 / 0111433, which is incorporated herein by reference.

[0044] Described herein are electrokinetic devices comprising:

[0045] a first substrate having a matrix of electrodes, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating comprising:

[0046] a dielectric layer in contact with the matrix electrodes,

[0047] a conformal layer in contact with the dielectric layer, and

[0048] a hydrophobic layer in contact with the conformal layer;

[0049] a second substrate comprising a top electrode;

[0050] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0051] a voltage source operably coupled to the matrix electrodes.

[0052] The electrokinetic devices as described can be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for introducing reagents as needed.

[0053] The devices can be used in any biochemical assay process involving high solute (ion) strength solutions, where high ion concentrations would otherwise degrade and impede the use of prior art devices. These devices are particularly advantageous for processes involving biomolecule synthesis, such as for example nucleic acid synthesis, for example using template independent strand elongation, or cell-free protein expression using different populations of nucleic acid templates.

[0054] FIGURES

[0055] Figure 1 shows a cross-sectional schematic of a conventional EWoD device;

[0056] Figure 2 A cross section of a device according to the application is shown;

[0057] Figure 3 A device according to the application is depicted with applied voltage and droplets;

[0058] Figure 4 An active matrix used in connection with the application is depicted;

[0059] Figure 5 A shows the deterioration of the array elements on a device without any conformal layer;

[0060] Figure 5 B shows an array of elements coated with parylene C and without any defects; and

[0061] Figure 6 The sequence of images representing the formation of droplets on a device according to the application is depicted.

[0062] Detailed description

[0063] Figure 1 A conventional electrowetting device is depicted with a substrate 10 and a plurality of individually controllable elements 11. The individually controllable elements can be arranged in an array so that a plurality of droplets can be manipulated simultaneously. The electrical properties of the individually controllable elements 11 can be different. For example, each individually controllable element can comprise an electrode or an electrical circuit. As shown, each individually controllable element is connected to a voltage source. Alternatively, each element can 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. Figure 1

[0064] On top of the individually controllable elements 11 is a dielectric layer 12. Instead of a dielectric layer 12, there can be an insulator. The insulator / dielectric can be made of SiO2, silicon oxynitride, Si3N4, 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 silicone, epoxy resin or mixtures thereof. The insulator / dielectric layer has a thickness of 10-10 000 nm.

[0065] On top of the insulator 12 (or dielectric) is a hydrophobic layer 13. The hydrophobic layer can comprise a fluoropolymer, such as Teflon, CYTOP or PTFE. The hydrophobic layer can be made of an amorphous fluoropolymer or a siloxane or organosilane. The hydrophobic layer has a thickness of 1-1000 nm.

[0066] A second electrode 14 is located opposite the array of individually controllable elements and the second electrode and the individually controllable elements are separated by a spacer 15 defining an electrically active workspace.

[0067] Figure 2 ​An electrowetting device according to the application is depicted, wherein on top of the individually controllable element is a functional coating, which comprises three components: a dielectric layer 12, a conformal layer 30 and a hydrophobic layer 13. According to an embodiment, the conformal coating is made of parylene or preferably parylene C. The conformal layer 30 has a thickness of 10-10000 nm and prevents ions from interacting with the insulator / dielectric layer 12. The second electrode 14 can comprise a second hydrophobic layer facing the (first) hydrophobic layer. An electrokinetic working space is then formed between the hydrophobic layers.

[0068] To facilitate adhesion between the different layers, often gaseous precursors are used. This can be used when depositing the layers using spin coating or dip coating.

[0069] A 1 M aqueous solution is applied to the substrate and a voltage is applied. As shown, by applying the voltage, the aqueous solution forms a droplet 35 over the individually controllable element. Figure 3

[0070] Figure 4 An array of individually controllable elements forming an electrode array 202 is depicted. Figure 4 is a diagrammatic view of an exemplary drive system 200 for controlling droplet operations by AM-EWoD propulsion of the electrode array 202. The AM-EWoD drive system 200 can be in the form of an integrated circuit attached to a support plate. The elements of the EWoD device are arranged in a matrix form with 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 the corresponding electrode, each TFT being connected to one of the gate lines and one of the data lines. The electrode of the element is represented as a capacitor Cp. A storage capacitor Cs is arranged in parallel with Cp and is not shown separately in Figure 4 .

[0071] The controller shown comprises a microcontroller 204, which includes control logic and switching logic. It receives input data from the input data lines 22 relating to the droplet operation to be performed. The microcontroller has an output for each data line of the EWoD matrix, providing a data signal. Data signal lines 206 connect 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 select signal. Gate signal lines 208 connect each output to a gate line of the matrix. A data line driver 210 and a gate line driver 212 are arranged in each data line and gate signal line respectively. The figure only shows the signal lines for those data lines and gate lines shown in the figure. The gate line driver can be integrated in a single integrated circuit. Similarly, the data line driver can be integrated in a single integrated circuit. The integrated circuit can include the complete gate driver assembly as well as the microcontroller.

[0072] ​Integrated circuits can be integrated onto the support board of the AM-EWoD device. These integrated circuits can be incorporated into the entire AM-EWoD device drive system.

[0073] The data line driver provides the signal level corresponding to the droplet operation. The gate line driver provides a signal for selecting the gate line whose electrode will be driven. The voltage sequence of one of the data line drivers 210 is... Figure 4 As shown in the image.

[0074] like Figure 4 As shown, a traditional AM-EWoD cell uses row-by-row addressing, where one gate line n is high and all other gate lines are low. Signals on all data lines are then transmitted to all pixels in the nth row. At the end of the row time, the gate line n signal goes low, and the next gate line n+1 goes high, allowing data from the next line to be transmitted to the TFT pixels in the n+1th row. This continues to scan all gate lines sequentially, thus driving the entire matrix. This is the same method used in almost all AM-LCDs such as mobile phone screens, laptop screens, and LC-TVs, as well as AM-EPDs (electrophoretic displays), whereby TFT control maintains the voltage on the liquid crystal layer.

[0075] Figure 5 A depicts an array of elements on an AM-EWoD device without a conformal layer. A driving voltage has been applied to a high ionic strength solution, and it can be seen that this results in damage and defects around some element edges. An example is highlighted in the dashed box. The result of this damage is the inability to drive aqueous droplets in the region using EWoD, further preventing the aqueous droplets from wetting the region, and / or generally also preventing the dispensing or splitting of existing aqueous droplets to form two droplets.

[0076] Figure 5 B shows something similar to Figure 5 The element arrays described in A, but coated with parylene C. Similarly, a driving voltage has been applied to the high ionic strength droplets, but it does not cause... Figure 5 The defect seen in A. The result of the conformal coating is no... Figure 5 The damage seen in A results in aqueous droplets being able to wet the area and / or be dispensed or split from existing droplets to form two droplets in the area of ​​the AM-EWoD device where high ionic strength droplets are in contact.

[0077] Experimental details

[0078] Adhesion promotion

[0079] Add 0.5% volume / volume of Silane A-174 to isopropyl alcohol / water in a 1 : 1 ratio and stir for 30 seconds to form Solution 1. Allow Solution 1 to sit for at least 2 hours to fully react and use within 24 hours. Submerge the substrate in Solution 1 for 30 minutes while ensuring the flexible ribbon of the TFT array remains dry. Remove the substrate and air dry for 15 minutes then clean in isopropyl alcohol for 15-30 seconds with tweezers under agitation. Dry the substrate with an air gun and store in a Teflon box for 30 hours prior to parylene C coating.

[0080] Parylene coating

[0081] Substrates (silanated and non-silanated) ready for coating were arranged face up on a clean glass slide next to a rotating stage inside a deposition chamber of a thoroughly cleaned SCS Labcoter 2 and the chamber was sealed. 50 mg of parylene C dimer was weighed into a disposable aluminum boat and loaded into the sublimation chamber. The system was sealed and evacuated to 50 mTorr before liquid nitrogen was added to the cold trap. The system was continuously evacuated throughout the deposition process. The sublimation chamber was heated to 175 °C with a heater cycle to maintain a target pressure of 0.1 Torr. The sublimation chamber was connected to the deposition chamber through a pyrolysis zone which was heated to 690 °C at a target pressure of 0.5 Torr. The deposition zone was maintained at ambient temperature, approximately 25 °C and approximately 50 mTorr. The system was held at temperature and pressure for two hours. The system was allowed to gradually return to ambient temperature over 30-40 minutes, then the stage and vacuum pump were turned off and the system was vented. The sample was removed from the deposition chamber and the coating thickness was verified to be approximately 100 nm by profilometry.

[0082] The device was then subjected to 22 hours of continuous operation with a high salt solution. Figure 6 Depiction of reliable droplet dispensing by electrowetting actuation even after 22 hours of continuous operation (from Figure 6 Dispensing electrowetting actuation shown in the top left to top middle to top right images, as opposed to the AM-EWoD device shown in A. Even after this, droplets can be moved over continuously actuated regions (as shown in the bottom left to bottom middle to bottom right images). Figure 5 Figure 6

[0083] Applications of the invention

[0084] The present invention can be used for a large number of different applications. In particular, the present invention can be used for moving cells, nucleic acids, nucleic acid templates, proteins, starting oligonucleotide sequences for nucleic acid synthesis, beads, magnetic beads, cells immobilized on magnetic beads, or biopolymers immobilized on magnetic beads.

[0085] ​​In these applications, the step of disposing aqueous droplets having ionic strength on the first matrix electrode and providing a differential potential can be repeated multiple times. They can be repeated more than 1000 times or more than 10,000 times, sometimes over a period of 24 hours.

[0086] The present method can be used for 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-reversible terminator 5'-triphosphate nucleosides to the 3'-terminus of a 5'- immobilized nucleic acid. During enzymatic nucleic acid synthesis, the following steps are taken on the instrument:

[0087] I. Bring an addition solution containing TdT, optionally pyrophosphatase (PPiase), 3'-O-reversible terminator dNTPs, and the desired buffer (containing salts and necessary reaction components such as metal divalent) to a reaction zone containing immobilized nucleic acid, where the nucleic acid is immobilized on a surface via a covalent bond to the 5' terminus of the nucleic acid, such as by magnetic beads. The initially immobilized nucleic acid can be referred to as a starting oligonucleotide and contains N nucleotides, for example 3-100 nucleotides, preferably 10-80 nucleotides, more preferably 20-65 nucleotides. The starting oligonucleotide can contain a cleavage site, such as a restriction site or a non-canonical DNA base such as U or 8-oxoG. The addition solution can optionally contain a phosphate sensor such as E. coli phosphate binding protein conjugated to a MDCC fluorophore to assess the quality of nucleic acid synthesis as a fluorescent output. The dNTPs can be combined in proportions to make a DNA library such as an NNK synthesis.

[0088] II. Apply a wash solution in the form of loose or discrete droplets to the reaction zone to wash away the addition solution. The wash solution generally has a high solute concentration (>1 M NaCl).

[0089] III. Apply a deprotection solution in the form of loose or discrete droplets to the reaction zone to deprotect the 3'-O-reversible terminator added to the immobilized nucleic acid in the immobilized nucleic acid zone in step I. The deprotection solution generally has a high solute concentration.

[0090] IV. Apply a wash solution in the form of loose or discrete droplets to the reaction zone to wash away the deprotection solution.

[0091] V. Repeat steps I-IV until the desired sequence is synthesized, for example repeat steps I-IV 10, 50, 100, 200, or 1000 times.

[0092] The method can be used to prepare oligonucleotide sequences via synthesis or assembly. The device allows for the definition of synthesis and movement of sequences. Using the method, starting sequences can be modified and extended oligonucleotides prepared at specific locations above the electrodes. Starting sequences at different locations can be exposed to different nucleotides, thereby synthesizing different sequences at different regions of the electrokinetic device.

[0093] After different defined sequence groups have been synthesized at different regions of the electrokinetic device, the sequences can be further assembled into longer contiguous sequences by linking two or more synthesized strands together.

[0094] Described herein is a method for preparing a contiguous oligonucleotide sequence of length at least 2n bases, the method comprising employing an electrokinetic device as described herein having a plurality of immobilized starting oligonucleotide sequences, wherein one or more comprises a cleavage site, using the starting oligonucleotide sequences to synthesize a plurality of immobilized oligonucleotide sequences of length at least n bases, using cycles of extension of reversibly blocked nucleotide monomers, selectively cleaving at least two immobilized oligonucleotide sequences of length at least n bases into the reaction solution while leaving one or more immobilized oligonucleotide sequences linked, hybridizing at least two cleaved oligonucleotides to each other to form a splint, and hybridizing one end of the splint to one of the immobilized oligonucleotide sequences and ligating at least one of the cleaved oligonucleotides to the immobilized oligonucleotide sequence, thereby preparing a contiguous oligonucleotide sequence of length at least 2n bases.

[0095] The synthesis and assembly steps can involve high solute concentrations, where ionic strength can degrade a device without a protective conformal layer.

[0096] The method of moving aqueous droplets can also be used to help facilitate cell-free expression of peptides or proteins. In particular, the method of the invention can be used to move droplets containing nucleic acid templates and cell-free systems with components for protein expression in an oil-filled environment in the electrokinetic device.

[0097] The invention can be used to automate the movement of droplets in a cartridge. For example, droplets intended for analysis can be moved in accordance with the invention. The invention can be incorporated into a cartridge for use in a local clinician diagnosis. For example, it can be used in conjunction with a nucleic acid amplification test (NAAT) to determine nucleic acid targets in, for example, a genetic test for an indication such as a cancer biomarker, a pathogen test to detect bacteria in, for example, a blood sample, or a viral test such as SARS-CoV-2 for coronavirus, for example for COVID-19 diagnosis.

[0098] The device can be thermocycled to effect nucleic acid amplification, or the device can be held at a desired temperature for isothermal amplification. Synthesis of different sequences in different regions of the device allows for multiplexed amplification using different primers in different regions of the device.

[0099] In addition, the present application can be used in conjunction with next generation sequencing, where DNA is synthesized by addition of nucleotides and a large number of samples are sequenced in parallel. The present application can be used to accurately locate individual samples used in next generation sequencing.

[0100] The present application can be used for automated library preparation for next generation sequencing. For example, the ligation step of sequencing adapters can be performed on the device. Then, selective subsets of sequences amplified from a sample can be ligated adapters so that the amplified population is sequenced.

[0101] As used herein, "and / or" shall be taken to mean either one or both of the items specifically associated with the term, with the conjunctive phrase acting as an abbreviation for the term "and / or." For example, "A and / or B" shall be interpreted to mean: (i) A, (ii) B, and (iii) A and B.

[0102] The description and definitions of the above-mentioned features are not limited to any particular aspect or embodiment of the application, unless the context indicates otherwise, and apply equally to all aspects and embodiments described.

[0103] Those skilled in the art will further appreciate that, although the present application has been described in terms of several embodiments, it is not limited to the embodiments disclosed. Rather, the present application is intended to cover alternatives, modifications and equivalents of the embodiments that are included within the scope of the present application as defined by the appended claims.

Claims

1. A method for moving aqueous droplets, the method comprising: An electric device is provided, the electric device comprising: 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: A dielectric layer comprising multiple layers of material, selected from silicon nitride, hafnium oxide, or aluminum oxide, is in contact with the matrix electrodes. A conformal layer containing parylene in contact with the dielectric layer, and A hydrophobic layer in contact with the conformal layer, wherein the parylene serves as a protective coating between the dielectric layer and the hydrophobic layer; A second substrate including a second electrode, wherein the second substrate further includes a second hydrophobic layer disposed on the second electrode; A spacer disposed between the first substrate and the second substrate and defining an electric working space; and A voltage source operatively coupled to the matrix electrodes; Aqueous droplets are provided on the first matrix electrode; and The voltage source provides a differential potential between the first matrix electrode and the second matrix electrode, thereby moving the aqueous droplet between the first matrix electrode and the second matrix electrode.

2. The method according to claim 1, wherein, The aqueous droplets have an ionic strength greater than 0.1 M.

3. The method according to claim 1, wherein, The aqueous droplets have an ionic strength greater than 1.0 M.

4. The method according to claim 1, wherein, The dielectric layer is 10 nm to 100 μm thick.

5. The method according to claim 4, wherein, The dielectric layer includes: A first layer comprising aluminum oxide or hafnium oxide, wherein the thickness of the first layer is 9 nm to 80 nm; A second layer comprising tantalum oxide or hafnium oxide, the second layer having a thickness of 40 nm to 250 nm; and A third layer comprising tantalum oxide or hafnium oxide, the third layer having a thickness of 5 nm to 60 nm, wherein the second layer is disposed between the first layer and the third layer.

6. The method according to claim 1, wherein, The conformal layer containing parylene is 100 nm thick.

7. The method according to claim 1, wherein, The hydrophobic layer includes a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite material, a zinc oxide polystyrene nanocomposite material, precipitated calcium carbonate, a carbon nanotube structure, a silica nanocoating, or a smooth liquid-filled porous coating.

8. The method according to claim 1, wherein, The functional coating includes a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer.

9. The method according to claim 1, wherein, The electric device also includes a controller to regulate the voltage supplied to a single matrix electrode.

10. The method according to claim 9, wherein, The electric actuator also includes multiple scan lines and multiple gate lines, wherein each of the thin-film transistors is coupled to the scan lines and the gate lines, and the multiple gate lines are operatively connected to the controller.

11. The method according to claim 1, wherein, The aqueous droplets have a volume of 1 μL or less.

12. The method according to any one of claims 1 to 11, further comprising: A second aqueous droplet is placed on the third matrix electrode; and A voltage source is used to provide a differential potential between the third matrix electrode and the second matrix electrode, thereby bringing the aqueous droplet into contact with the second aqueous droplet.

13. A method for performing droplet-based nucleic acid synthesis, droplet-based nucleic acid amplification, or droplet-based nucleic acid assembly, wherein, The method includes repeating the method of any one of claims 1 to 12 to add a nucleotide to a starting oligonucleotide or to link two or more nucleic acid chains in one or more droplets.

14. A method for performing cell-free expression of peptides or proteins based on droplets, wherein, The method comprises repeating the method of any one of claims 1 to 12, wherein the droplet contains a nucleic acid template and a cell-free system having protein expression components.

15. The method of claim 13 or 14, wherein, The aqueous droplets moved between the first matrix electrode and the second matrix electrode more than 1000 times.

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