Controlled reservoir filling

Abstract

There is provided herein a method for controlled filling of a plurality of reservoirs on a microfluidic device with a defined area of fluid, the method comprising: a. Providing a device having a plurality of apertures connected via an inlet to a substrate with a plurality of electrodes; b. Filling the device with a filling liquid that is immiscible with water such that the plurality of holes are at least partially filled with the filling liquid; c. Loading two or more of the apertures with the aqueous liquid falling into the apertures; and d, extracting a part of the filling liquid from the device so as to suck the aqueous liquid onto the plurality of electrodes through the inlet to form a plurality of aqueous liquids with different sizes and volumes.

Description

Field of the Invention

[0001] Provided herein are methods for achieving automated bubble-free loading of an aqueous phase reservoir covering more than one electrode on a microfluidic device having a defined fluid area. The present invention is in the field of electrowetting on fluid dielectrics (EWoD) and devices utilizing these phenomena. Background of the Invention

[0003] Microfluidic devices for manipulating droplets or particles based on electrical signals have been widely described. Electrokinesis (movement due to an electrical signal) occurs due to (1) an inhomogeneous electric field affecting the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or (2) a change in the contact angle of the liquid on a solid surface (electrowetting on dielectrics or EWoD). DEP can also be used to generate forces on polarizable particles to induce their movement. Electrical signals can be transmitted to discrete electrodes, transistors, transistor arrays, or semiconductor membranes, whose electrical properties can be modulated by optical signals.

[0004] Manipulation of droplets by applying an electric potential can be achieved on electrodes covered with an insulator or dielectric or a series of insulators or dielectrics. Manipulation of droplets caused by an applied electric potential is called electrowetting. In the case of droplets in a channel, 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 a barrel wall or microfluidic tube. Embedded in the barrel wall or tube wall are electrodes covered by a dielectric layer, each of which is connected to a circuit that can be quickly turned on and off at intervals to change the wetting properties of the droplets on the dielectric layer. This creates the ability to guide droplets along a given path.

[0005] Compared to channel-based microfluidics, digital microfluidics (DMF) uses alternating current signals on an electrode array to move fluids on the array surface. Therefore, liquids can be moved on open devices through electrowetting. Digital microfluidics enables precise control of droplet operations, including droplet movement, fusion, and separation.

[0006] DMF exploits the EWoD phenomenon when a droplet is actuated between two parallel electrodes (forming a cell gap) covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in 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:

[0007]

[0008] in, is the contact angle when the electric field across the interfacial layer is zero, is the liquid-gas tension, c is the specific capacitance (as Given, where ε r is the dielectric constant of the insulator / dielectric, is the permittivity of a vacuum, t is the thickness), and V is the applied voltage or potential. Therefore, the change in contact angle (induced droplet movement) is a function of surface tension, potential, dielectric thickness, and dielectric constant.

[0009] The planar DMF device consists of two substrates separated by a cell gap, which is typically a few hundred micrometers. In this cell gap, an electric potential is applied to manipulate aqueous droplets by changing the surface wettability by actuating electrodes. For practical applications, reagents / samples will be loaded into this cell gap to form a gap reservoir, from which smaller sub-droplets are then dispensed.

[0010] When a droplet is actuated by an EWoD, two sets of opposing forces act on it: the electrowetting force induced by the electric field and the drag force, which consists of the drag force caused by the interaction of the droplet with the filler dielectric and the contact line friction. The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all the drag forces is determined by the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε). r ) 1 / 2 Therefore, in order to reduce the actuation voltage, it is necessary to reduce (t / ε r ) 1 / 2 (i.e. increasing the dielectric constant or decreasing the thickness of the insulator / dielectric). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, depositing 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 actuate the droplets. Therefore, most academic studies report using much higher voltages of >100V on easily fabricated thick dielectric films (>3 microns) to achieve electrowetting.

[0011] However, high voltage EWoD-based devices with thick dielectric films have limited industrial applicability, primarily due to their limited droplet multiplexing capabilities. The use of low voltage devices including thin film transistors (TFTs) and optically activated amorphous silicon layers (a-Si) has paved the way for industrial applications of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplexed manner. The actuation voltage of TFTs or optically activated a-Si is low (typically <15V). The bottleneck in the fabrication of low voltage devices and, therefore, the adoption of 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.

[0012] EWoD uses electric fields to manipulate droplets and perform droplet operations such as moving, mixing, and splitting. Droplets are typically generated from an interstitial reservoir formed in the cell gap (defined by a spacer introduced between two electrically addressable substrates) that is metered via a fluid applicator such as a pipette or an automated fluid delivery subsystem.

[0013] It is critical to load the metered area in a bubble-free manner. DMF devices have a low surface energy coating within the cell gap; this, together with the relatively hydrophilic plastic housings on the ports, makes loading difficult. These plastic housings tend to draw liquid out of the EWoD, back to fill the plastic ports rather than the interstitial reservoirs in the cell gap. During loading, bubbles are easily introduced, which prevent the reservoir from filling and affect its function. Pressurizing the liquid in order to force entry often results in the liquid being pressed back out of the entry port when the pressure is released.

[0014] There are many prior art methods for loading EWoD devices, but the prior art methods are not optimal. For example, US2015352544 describes a system configured to perform a specified reaction for biological or chemical analysis. The system includes a liquid exchange component, which includes an assay reservoir for containing a first liquid, a receiving chamber for containing a second liquid immiscible with the first liquid, and an exchange port fluidly connecting the assay reservoir and the receiving chamber. The system also includes a pressure activator operably coupled to the assay reservoir of the liquid exchange component. The pressure activator is configured to repeatedly exchange the first and second liquids by (a) causing a specified volume of the first liquid to flow into the receiving chamber through the exchange port and (b) causing a specified volume of the second liquid to flow into the assay reservoir through the exchange port. The system also includes a fluid system in fluid communication with the liquid exchange component.

[0015] US2014 / 0008222 describes a method for aspirating liquid into a device using a syringe. As shown in, for example, FIG9 shows that liquid in a single reservoir can be aspirated into the device by withdrawing the same amount of liquid from the opposite reservoir. FIG9 shows only a single reagent in the loading port.

[0016] GB2542372 describes a passive loading method based on pressure balance. A volume of reagent loaded into a well naturally displaces the venting fluid (air or fill fluid) in the device, due only to the hydrostatic head pressure caused by the additional loaded fluid.

[0017] US2020108396 describes a microfluidic device comprising a substrate spaced apart between an upper and a lower portion defining a fluid chamber therebetween; a hole for introducing a fluid into the fluid chamber; and a fluid input structure disposed above the upper substrate, having a fluid hole for receiving a fluid from a fluid applicator inserted into the fluid hole. The fluid hole is connected to a fluid outlet disposed in a base of the fluid input structure, and the fluid outlet is adjacent to the hole. The fluid hole includes a first, a second, and a third portion, wherein the first portion of the hole forms a reservoir filled with fluid; the second portion of the hole is configured to fit well with an outer surface of a fluid applicator inserted into the fluid hole. The third portion of the hole is connected to the fluid outlet, and the diameter at the interface between the third portion and the second portion is greater than the diameter of the second portion at the interface between the third portion and the second portion.

[0018] It is therefore an object of the present invention to provide an improved method for loading multiple aqueous liquid areas into an EWoD device. The present invention enables simultaneous loading of multiple reservoirs of different sizes. The present invention enables simultaneous loading of multiple reservoirs from different sides of the device. Any operation on a DMF-based device requires accurate measurement of droplets and calibration to compensate for variability in the manufacturing technology. For example, in order to accurately dispense droplets from a reservoir held on the device, the filling area of ​​the reservoir needs to be accurately controlled. The reservoir volume is a function of the cell gap height and the actuation area of ​​the DMF filled during loading. Therefore, the volume is defined by the area, which in turn is defined by the number of actuated pixels. Variations in the gap height and the interaction between the aqueous reagents and the loading ports make it difficult to achieve a consistent area for loading. For applications where reagents are loaded at regular intervals to run cycles, loading errors can lead to incorrect reservoir filling. For AM-EWoD (active matrix electrowetting on dielectric) DMFs, the high-resolution active matrix on the backplane of the TFT increases the complexity of reservoir loading because the user (or algorithm) cannot easily determine the level to which the reservoir must be filled. Conventional passive EWoD devices have discrete reservoir electrodes that make filling them easier by providing visual and / or electronic feedback.

[0019] The planar DMF device consists of two substrates separated by a cell gap of typically several hundred microns. In the cell gap, an electric potential is applied to manipulate aqueous droplets by changing the surface wettability by actuating electrodes. For practical applications, reagents / samples will be loaded into the cell gap to form a gap reservoir, from which smaller sub-droplets are then dispensed. Droplets can be obtained by repeatedly dividing the reservoir or by repeating the dispensing operation. The success rate, distribution accuracy, and distribution precision of dispensing sub-droplets all depend on the reservoir area that meets the expectations. It is difficult to control reservoir filling by metering the exact volume entering the cell gap, including the difficulty of cell gap variation leading to underfilling or overfilling, and the Teflon on the DMF device. TMAnd / or the hydrophobicity of the plastic shell generates capillary forces. Such problems are particularly serious when multiple reservoirs are loaded at the same time.

[0020] US 7,763,471 describes a fluidic device loaded with reagents on a portion of the chip external to the device, ie the device contains wells separate from the DMF.

[0021] US 8,821,705 shows a digital microfluidics system equipped with a single pipette.

[0022] US2011 / 0220505 describes a method for transferring liquids on an EWoD device that uses only actuation to initiate liquid flow between reservoirs on the device. Actuation can be performed using multiple channels ("tooth electrodes"). The teeth are used to move and direct the liquid. SUMMARY OF THE INVENTION

[0024] A method for controlling the filling of a reservoir on a microfluidic device is provided herein. More specifically, a method for controlled filling of a reservoir on an electrowetting on dielectric (EWoD) device. More specifically, a method for simultaneously loading multiple reservoirs of different capacities without introducing air through an inlet port.

[0025] The loading of reagents is a function of several interdependent processes; EWoD forces drawing reagents into (and retaining) the capillary forces drawing reagents into the plastic ports, fluctuations within the cell gap during pressure driven loading; the surface tension of the reagents and the Laplace pressure defined by the shape of the reagents in the plastic ports and the cell gap. Typically, the interior surfaces of the device are more hydrophobic than the surfaces of the loading device. The present invention relates to improved methods for loading a variety of aqueous reagents with greater accuracy of target and metered volumes. Thus, the proposed methods are particularly advantageous for loading reagents with high surface tension and for designing fluid delivery enclosures using plastics that are generally hydrophilic.

[0026] A method of loading an aqueous liquid into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed, the method comprising:

[0027] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0028] b. filling the device with a water-immiscible filling liquid, such that the plurality of holes are at least partially filled with the filling liquid;

[0029] c. loading one or more of the holes with an aqueous liquid dropped into the hole; and

[0030] d. A portion of the fill solution is withdrawn from the device, thereby drawing aqueous liquid through the inlet port onto the multiple electrodes.

[0031] A method for loading a plurality of different sized volumes of aqueous liquid into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed, the method comprising:

[0032] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0033] b. filling the device with a water-immiscible filling liquid, such that the plurality of holes are at least partially filled with the filling liquid;

[0034] c. loading one or more of the holes with an aqueous liquid dropped into the hole; and

[0035] d. A portion of the filling liquid is withdrawn from the device, thereby drawing the aqueous liquid onto the plurality of electrodes through the inlet to form a plurality of volumes of aqueous liquid of different sizes.

[0036] The volume and flow direction of the introduced aqueous liquid can be controlled using electrodes on the substrate.

[0037] The device may be an active matrix thin film transistor (AM-TFT) based device.

[0038] Also disclosed is an active matrix thin film transistor (AM-TFT) device having a substrate with a plurality of electrodes, the device comprising a plurality of fluid inlet ports on at least two sides of the device, wherein the inlet ports on each side of the device are evenly spaced, and wherein the device is connected to a syringe pump.

[0039] The device may include two substrates, wherein at least one substrate has a plurality of electrodes, and the two substrates define parallel plates separated by a spacer to define a volume.

[0040] The device can be made of a bottom plate with an electrode array (TFT), a top plate (optionally glass, although it can be plastic) defining a cell gap therebetween. The top plate can contain holes for loading reagents, or the top and bottom plates can be sealed with a spacer with an inlet. The inlet can be located below a housing (e.g., made of plastic) containing an entry loading port to load reagents into the cell gap. The device is filled with a filling fluid to fill the cell gap and at least partially fill all entry ports. Using an external source, such as a multichannel pipette, the reagent is placed into the port. The reagent sinks to the bottom end of the port, near the inlet in the top glass. The filling fluid is removed from the cell gap and the reagent is introduced into the device for downstream electrowetting operation.

[0041] The device is connected so that the fill fluid level within the device can be adjusted. One or more ports are connected to a fill fluid source. The loading process can be driven by removing fill fluid from one port or from multiple ports simultaneously. Multiple ports can be placed on the same side or opposite sides of the device. Oil can be removed from two corners of the device, which can be located on the same side or diagonal corners. Many ports on the device can be loaded simultaneously by simply removing fill fluid from the cell gap. The entry path taken by the aqueous liquid can be controlled by electrodes on the array. Electrode activation alone is not sufficient to draw liquid from the hydrophilic port to the hydrophobic cell gap, and fill fluid is required to flow from the cell gap. The fill fluid should be retained in the entry port to prevent air from entering the device through the entry port.

[0042] The filling liquid can be a hydrophobic or non-ionic liquid. For example, the filling liquid can be decane or dodecane. The filling fluid can be a silicone oil, such as dodecamethylpentasiloxane (DMPS). The filling liquid can contain a surfactant, such as a sorbitan ester, such as Span 85.

[0043] The fluid inlet can be via a hole in the upper plate or through a spacer. The inlet can be in the top substrate. Multiple electrodes can be on the bottom substrate. The top substrate is made of glass or polymer and can range in thickness from 0.5 mm to 20 mm.

[0044] The wells / entry ports can be designed to avoid angle variations where aqueous liquids can be trapped. Thus, the sides of the wells can form a continuous curve without inflection points. Thus, heavier liquids introduced into the wells sink to the bottom of the wells regardless of the volume added to the wells or the size of the wells. The lack of inflection points prevents reagents from being trapped and reduces the entry of air bubbles.

[0045] The spacer may comprise an adhesive having beads of a defined size distribution. The spacer may comprise a polymer material of a defined thickness. The spacer may comprise glass, in which case the layers may be fused together. The spacer gap, and therefore the fluid height in the device, may be 50 microns to 250 microns. The spacer gap, and therefore the fluid height in the device, may be 100 microns to 150 microns. The spacer may comprise tape, which may be an adhesive.

[0046] The fill fluid may be moved via automated means, or may be moved under gravity. A hydrostatic head may be used to move the liquid within the device. Prior to loading the aqueous reagent, the wells are at least partially filled with a fill fluid. The fill fluid may be less dense than the aqueous phase, so that the aqueous phase sinks into the wells. Alternatively, the aqueous phase may be located above the fill fluid, in which case all of the fill fluid must be pumped out of the wells to allow the aqueous fluid to enter.

[0047] When multiple volumes of reagent are to be loaded, the liquid is typically moved by withdrawing fill fluid. Some or all of the aqueous reagent is introduced from each well to which the aqueous reagent has been added. Since each well contains fill fluid, uniform pressure is maintained as the fill fluid is withdrawn. Once all of the aqueous reagent has been drawn into the device, fill fluid located above the device can enter, thereby preventing any air from being drawn into the device. In the event that fill fluid is not present in the entry port, either a large amount of aqueous liquid is required to fill the port, or once all of the aqueous liquid has been drawn out, the port with the smallest volume of aqueous reagent added allows air to enter the device.

[0048] The device can be connected to a pump, such as a syringe pump, a peristaltic pump, a disc pump, a diaphragm pump or a pneumatic pump. The pump enables the device to be filled with a filling liquid in an automated manner. Once filled, the pump enables partial withdrawal of the filling fluid to create a negative pressure in the device, which draws the reagent from the hole. Thus, the filling and withdrawal of the fluid can be performed in an automated manner to allow for a largely "hands-free" loading of aqueous reagents. The automated filling liquid filling and withdrawal method can be integrated into an instrument that provides other functions associated with a digital microfluidic device, including heating, cooling, optical, sensing, mechanical and magnetic functions.

[0049] The inlets may be 90 degrees from each other. The inlets may be 180 degrees from each other. The inlets may be on 4 sides of the device. Each side may have at least 4, 8 or 12 ports. Each side may have 8 ports. The device may have 4 groups of 8 ports. The number of ports may be different on different sides of the device, for example, one side may have 8 ports and one side may have 4 ports. The device may have 8 ports on 3 sides and 16 ports on the fourth side. The device may have 8 ports on 2 sides and 16 ports on 2 sides. The device may have 8 ports on 1 side and 16 ports on 3 sides. The device may have 16 ports on 4 sides. The ports may be offset to provide multiple rows of linear ports on one side, such as a first row and a second row, wherein the second row is offset backward from the first row so that the source liquid can flow between the ports in the first row. The rows may be zigzag. Multiple volumes may be loaded from opposite faces. For example, at least 32 volumes of aqueous liquid having at least 2 different volumes may be formed. At least 8 volumes can be loaded simultaneously from 4 opposing sides.

[0050] The device can be loaded evenly from multiple sides by withdrawing the filling fluid. The device can be loaded from 4 sides. The filling fluid can be extracted from one or more corners of the cell gap. An automated syringe pump can be used to remove the filling fluid from the cell gap through two ports at different corners of the device.

[0051] The ports can be located in a housing, such as a plastic housing adjacent to the entrance to the cell gap. The spacing between the entry ports can be 9 mm. The spacing between the entry ports can be 4.5 mm. The spacing of the entry ports is 4.5 mm or a multiple thereof. This will cover 24-hole, 48-hole, 96-hole, 384-hole ports. The spacing of the ports can be the same on each side of the device, or can be of different sizes. In this case, the spacing refers to the distance between the centers of each entrance.

[0052] The volume of aqueous reagent loaded into each entry port can range from 1 microliter to 50 microliters. The volume can range from 1 microliter to 20 microliters. Different volumes can be loaded from each port.

[0053] The device and reagent can be used for protein expression. Therefore, the reagent loaded from the first side can contain a reagent for expressing protein, which can be a cell-free protein synthesis reagent. The reagent loaded from the second side can contain a nucleic acid template, which can be a linear or circular template. The reagent introduced from the first side can be merged with the reagent introduced from the second side. The merged reagent can achieve protein production. The reagent from the third side can achieve detection or purification of the expressed protein. The reagent can also include, for example, an additive that can achieve post-translational modification.

[0054] Aqueous liquid can be introduced into the hole by a pipette, a multichannel pipette, a syringe, a blister pack, an acoustic dispenser or a robotic liquid handler. Aqueous liquid can be loaded from multiple holes simultaneously, and the holes can be on the same side or multiple sides of the device. Each hole is a separate liquid and can be the same or different from the content of the aqueous volume in other holes. The volume of aqueous liquid loaded in each port can be the same or different.

[0055] Automatic filling and / or withdrawal of fill fluid may be software controlled.The device may be part of a larger instrumentation system that provides environmental control, such as temperature control or light control, and may have analytical capabilities, such as part of an optical system for fluorescence or luminescence assay detection.

[0056] The position of the aqueous layer is controlled by the actuation of the electrodes to form a reservoir in a defined area. Once the aqueous liquid is drawn onto the substrate with the plurality of electrodes, the plurality of electrodes are actuated to control the position of the aqueous liquid. Multiple reservoirs may be formed on the device. The reservoirs may contain different volumes of liquid, controlled by the number of electrodes actuated.

[0057] The device can be used for bioassay or synthesis of biopolymers. The loaded aqueous liquid can be selected from, for example, cell-free protein synthesis reagents, nucleic acid construct solutions, detection protein solutions, slurries of magnetic or superparamagnetic beads, buffer solutions in the pH range of 6-8, washing solutions, recombinant protein solutions, solutions of small molecules with molecular weights of 200 to 1000 g / mol.

[0058] The device can be used for cell-free protein synthesis. The cell-free protein synthesis reagent can be derived from an organism or formed by recombinant protein elements. The cell-free protein synthesis reagent can be a cell lysate or can be prepared from purified protein. The cell-free protein synthesis reagent can be a blend of cell lysate and purified protein. The cell-free protein synthesis reagent can be derived from prokaryotes, mammals, plants or insect organisms, such as Escherichia coli, H sapiens, C griseus, N tabacum or T aestivum.

[0059] Each hole may have one or more entrances. Each hole may have a single entrance to the substrate. The diameter of the entrance may be 0.2 mm to 2 mm. The diameter of the entrance may be 1 mm.

[0060] The method enables loading of the device without introducing bubbles and does not require positive external pressure. During loading, the substrate is always covered with fluid. Filling and / or removal of the filling fluid can be performed at a rate of 0.02 mL / min to 2 mL / min. Filling can be performed at 0.5 to 1 mL / min. Extraction can be performed at 0.05 mL / min to 0.1 mL / min.

[0061] Each reservoir can include X times Y pixels, where X is the width of 10-50 pixel range, and Y is the length of 10-150 pixel range. The size of the reservoir can be different, depending on the required volume to be further distributed from the reservoir. Reservoirs of different sizes can be formed simultaneously, for example, 8 or more reservoirs are formed at one time. By shifting out a portion of the filling fluid, the reagent loaded into each port can enter the device. Therefore, the time saved in an automated operation rather than continuously loading, for example, 40 reservoirs is a lot.

[0062] The time for loading using the manual method (pipetting) is related to the volume being loaded and the EW force. Multiple reservoirs take e.g. 30-50 minutes (user dependent time) for loading, whereas an automated syringe pump is about 8 minutes and does not require the user to handle the liquid after loading the well.

[0063] The method can be applied to loading reservoirs. By introducing visual calibration structures on the gap reservoir of the device, visual signals are generated, which help to accurately load the reservoir with a limited liquid area. These calibration structures are formed during the loading cycle of the reagent and merged into the reservoir during the distribution cycle. In fact, the calibration structure is an electrode temporarily actuated on the side of the reservoir electrode away from the fluid inlet. Once these temporary instantaneous areas are filled, the electrode forming the calibration structure is closed and liquid is added to the main reservoir. This visual signal can be automatically detected by computer vision software to achieve fully automatic and accurate loading.

[0064] A method for dynamically controlling the shape of an aqueous phase for controlled filling of a reservoir on an electrowetting on a planar dielectric (EWoD) device is disclosed, the method comprising:

[0065] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0066] b. Filling the device with a water-immiscible filling liquid such that the plurality of holes are at least partially filled with the filling liquid;

[0067] c. one or more of the holes are loaded with an aqueous liquid dropped into the hole;

[0068] d. withdrawing a portion of the filling fluid from the device, thereby drawing the aqueous liquid onto the plurality of electrodes through the inlet;

[0069] e. actuating the reservoir electrode to form a defined aqueous liquid reservoir on the device; and

[0070] f. Temporarily actuating electrodes on the side of the reservoir opposite the source aqueous liquid to form one or more visual calibration structures, which are the areas that fill last, so that when the temporarily actuated electrodes are closed, the liquid becomes part of the reservoir, thereby precisely controlling the liquid area in the reservoir.

[0071] The visual calibration structure can be an elongated protrusion. Each reservoir can have more than one elongated protrusion. Each reservoir can have two or three elongated protrusions. Each elongated protrusion can occupy 1% to 10% of the reservoir area.

[0072] The opposing sides may be at 90 degrees or 180 degrees to the source of flowing liquid entering the reservoir.The vision alignment structure may be located on more than one opposing side of the reservoir inlet. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Image showing aqueous fluid being loaded into a digital microfluidic device located in the instrument. Aqueous fluid loading is achieved by the presence of an automated syringe pump that can add or remove fill fluid at programmable defined flow rates.

[0075] Figure 2 Image showing a digital microfluidic device with multiple aqueous reservoirs loaded. Two sides of the device (right; bottom) are loaded with 8 reservoirs each, the third side (top) is loaded with 16 reservoirs, and the fourth side (left) is loaded with 4 reservoirs. Each reservoir can be of different sizes and dimensions, controlled by the volume of aqueous fluid loaded into each well and the pattern of electrodes actuated on the array.

[0076] Figure 3 show Figure 3 A digital microfluidic device as described in , which is in the process of being loaded with aqueous fluid. Once all aqueous fluids have been loaded into the appropriate wells and the filling fluid is started to be drawn out, loading occurs simultaneously from each side of the device.

[0077] Figure 4 A series of images (1-3) are shown demonstrating the formation of eight aqueous reservoirs with calibration structures driven by an air displacement multichannel pipette. Arrows show the formation of calibration structures on the reservoirs. The volume of the aqueous phase loaded was 5 μL, including both the reservoir to be formed and the calibration structures. The actuation area size of the main reservoir was 30 x 28 pixels, and each calibration structure was 6 x 6 pixels. The time required to fill the reservoir was 120 seconds. The aqueous reagent loaded was 0.05% weight / weight Pluronic F127 in aqueous buffer, which contained red food coloring to aid visualization (1: 1 dilution). The filling fluid in the device was 0.1% span85 in dodecamethylpentasiloxane (DMPS). Image (4) shows a snapshot of the electro-actuation pattern sent to the electrodes on the device during the reservoir filling process, where white represents the electrode to which the potential is applied. The calibration structure is shown by the arrow on the image. Figure 4 Image (1) shows the DMF device filled with filler fluid. Figure 4 Image (2) shows the initial stage of reservoir loading. Figure 4 Image (3) shows that two reservoirs have been filled to the correct volume (both calibration structures are visible), while the other reservoirs are still in the process of being formed on the device.

[0078] Figure 5aSchematic diagram showing the loading process (single port shown). The device is made of a bottom plate with an electrode array (TFT), a top plate (optionally glass, although it can be plastic) defining a cell gap therebetween. The top glass contains holes for loading reagents. The top glass holes are located beneath a plastic housing that contains access loading ports to load reagents into the cell gap. (A) The device is filled with filling fluid to fill the cell gap and at least partially fill all access ports. (B) Reagents are placed into the ports using an external source, such as a multichannel pipette (one channel shown). (C) Reagents sink to the bottom end of the ports, near the inlet in the top glass. (D) Filling fluid is removed from the cell gap and reagents are introduced into the device for downstream operations.

[0079] Figure 5b A schematic diagram showing a device with one port connected to a fill fluid extraction unit, multiple ports can be used to load aqueous reagents. The loading process can be driven by removing fill fluid from one or more ports. Multiple ports can be placed on the same side of the device or on opposite sides. Oil can be removed from two corners of the device, which can be on the same side or diagonally opposite corners.

[0080] Figure 6 Port geometry is shown to improve loading consistency. The lower image shows an improved design without an inflection point in the side wall. In the upper design, the aqueous reagent can be captured on a more horizontal area of ​​the wall, which can act as a baffle to prevent aqueous reagent ingress and increase bubble ingestion.

[0081] Figure 7 Shows loading of different volumes into multiple different reservoirs. The larger reservoir is 12 μL and the smaller reservoir is 3 μl. Loading occurs simultaneously with the withdrawal of fill fluid. This image shows loading in progress.

[0082] Figure 8 show Figure 7 The activation mode of the mount. DETAILED DESCRIPTION OF THE INVENTION

[0084] A method of loading an aqueous liquid into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed by pumping out a fill fluid to create a negative pressure that pulls the aqueous liquid onto the device. The negative pressure enables bubble-free loading and loading without having to apply positive pressure from an external source, which often causes the device to leak or blow out once the positive pressure is released.

[0085] A method of loading an aqueous liquid into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed, the method comprising:

[0086] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0087] b. Filling the device with a water-immiscible filling liquid such that the plurality of holes are at least partially filled with the filling liquid;

[0088] c. loading one or more of the wells with an aqueous liquid; and

[0089] d. A portion of the fill solution is withdrawn from the device, thereby drawing aqueous liquid through the inlet port onto the multiple electrodes.

[0090] A method of loading an aqueous liquid into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed, the method comprising:

[0091] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0092] b. Filling the device with a water-immiscible filling liquid such that the plurality of holes are at least partially filled with the filling liquid;

[0093] c. loading one or more of the holes with an aqueous liquid dropped into the hole; and

[0094] d. A portion of the fill solution is withdrawn from the device, thereby drawing aqueous liquid through the inlet port onto the multiple electrodes.

[0095] A method for loading a plurality of volumes of aqueous liquids of different sizes into a digital microfluidic device having a substrate with a plurality of electrodes is disclosed, the method comprising:

[0096] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0097] b. Filling the device with a water-immiscible filling liquid such that the plurality of holes are at least partially filled with the filling liquid;

[0098] c. loading two or more of the wells with an aqueous liquid dropped into the wells; and

[0099] d. withdrawing a portion of the filling liquid from the device, thereby drawing the aqueous liquid onto the plurality of electrodes through the inlet to form a plurality of volumes of aqueous liquid of different sizes.

[0100] A method for dynamically controlling the shape of an aqueous phase to controllably fill a reservoir on an electrowetting on a planar dielectric (EWoD) device is disclosed, the method comprising:

[0101] a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet;

[0102] b. Filling the device with a water-immiscible filling liquid such that the plurality of holes are at least partially filled with the filling liquid;

[0103] c. loading one or more of the wells with an aqueous liquid dropped into the wells; and

[0104] d. withdrawing a portion of the filling liquid from the device, thereby drawing the aqueous liquid onto the plurality of electrodes through the inlet;

[0105] e. actuating the reservoir electrode to form a defined reservoir of aqueous liquid on the device; and

[0106] f. Temporarily actuating electrodes on the side of the reservoir opposite the source aqueous liquid to form one or more visual calibration structures, which are the areas that fill last, so that when the temporarily actuated electrodes are closed, the liquid becomes part of the reservoir, thereby precisely controlling the liquid area in the reservoir.

[0107] The aqueous liquid may come from an external source via a pipette, multichannel pipette, syringe, blister pack, acoustic dispenser, or robotic liquid handler.

[0108] Automatic filling and / or withdrawal of fill fluid may be software controlled.The device may be part of a larger system providing environmental control such as temperature control, light control and may have analytical capabilities, such as part of an optical system for fluorescence or luminescence assay detection.

[0109] The formation of one or more visual calibration structures enables more accurate filling of the reservoir and therefore more standardized droplet sizes dispensed from the reservoir on the EWoD device. These calibration structures are elongated protrusions ("fork-like" structures) from the reservoir of the EWoD device that are the areas where the external reagent source is last to be filled.

[0110] The visual calibration structure is formed by electrodes on the actuation device, and the internal liquid is held in place by the electrode actuation to form an elongated protrusion. The number of electrodes activated to form the width of the calibration structure is less than the width of the defined reservoir. The number of electrodes activated to form the width of the calibration structure can be less than half the number of electrodes forming the width of the defined reservoir.

[0111] Liquid can enter through the top or side of the digital microfluidic array. The top entry point can be through a hole in the planar surface. The side entry can be through a gap in the adhesive holding the two planar surfaces together, or through a gap in the spacer material that defines the cell gap. The entry port can be formed by a hole in the upper surface or side of the planar EWoD device. The diameter of the hole can be about 1 mm.

[0112] The electrode array may be formed on a surface of the planar EWoD device opposite the surface having the inlet.

[0113] The external source may take the form of a pipette or a delivery tube. The pipette may be mechanical or electronic. The pipette may be single channel or multi-channel. A multi-channel pipette may have 8 channels or 12 channels. A method of loading an EWoD device using a multi-channel pipette, such as a pipette having 4, 8 or 12 channels, is disclosed herein.

[0114] The spacing between the entry ports can be 9 mm. The spacing between the entry ports can be 4.5 mm. The spacing of the entry ports is 4.5 mm or a multiple thereof. This will cover 24-hole, 48-hole, 96-hole, 384-hole ports. The spacing of the ports can be the same on each side of the device, or can be different sizes. In this case, the spacing refers to the distance between the centers of each inlet.

[0115] The inlet may be located in the top plate of the DMF device. The port may be a hole in the side of the fluid gap of the DMF device.

[0116] The inlet port can be tapered to prevent the loading process from damaging the electrodes on the device via physical contact. For example, when a multichannel pipette is placed in the inlet port, the taper prevents contact between the end of the pipette tip and the surface of the device.

[0117] The inlet port may be angled to prevent the loading process from damaging the electrodes on the device via physical contact. For example, when a multichannel pipette is placed in the inlet port, the angle prevents contact between the end of the pipette tip and the surface of the device. The angle is relative to the electrode array and is such that the inlet port is not 90 degrees relative to the array (horizontally). o (vertical). The loading device need not actually touch the access port as the loading liquid sinks into the well. The well contains the fill fluid and the added heavier aqueous liquid sinks into the well.

[0118] This paper discloses the use of a multichannel pipette to load the DMF device. The multichannel pipette can be mechanical, electronic or connected to a liquid handling robot arm. The multichannel pipette can have 4, 8 or 12 channels. The multichannel pipette can be a positive displacement pipette, an air displacement pipette or a liquid displacement pipette.

[0119] Disclosed herein is the use of a contactless acoustic processing dispensing device to load the port of a DMF device.

[0120] The volume of reagent loaded per inlet port can be from 1 microliter to 50 microliters. The volume can be from 1 microliter to 20 microliters.

[0121] Electrode actuation to form a temporary loading protrusion may occur over a period of time greater than 1 second. Electrode actuation may occur over a period of time of 10-120 seconds.

[0122] A visible delivery path can be used, using temporary electrode actuation to form a temporary hydrophilic path on an otherwise hydrophobic surface to improve the introduction of fluid into the reservoir. When the reagent is delivered to the reservoir, the path can use electrode actuation to form a temporary flow path between the entrance and the actuated reservoir. The temporary delivery path can be formed by actuating more than 2 electrodes. The delivery path can be formed by actuating 10-500 electrodes arranged in an elongated pattern. The delivery path can be formed by actuating electrodes arranged in an elongated pattern of 35 lengths and 8 widths. The delivery path can be formed by actuating electrodes arranged in an elongated pattern of 22 electrodes long and 4 electrodes wide. The pattern can be 22-35 electrodes long and 4-8 electrodes wide.

[0123] The on-chip reservoir can be formed at 2-500 electrodes from the access port. The on-chip reservoir can be formed with 0.1 to 100 μL. Multiple on-chip reservoirs can be formed using a single access port. Alternatively, multiple access ports can be used to combine reagents into one or more on-chip reservoirs.

[0124] Any electrokinetic method may be used for droplet release from a reservoir. Droplets may be moved using electrowetting on dielectrics (EWoD). The electrical signal on an EWoD or optical EWoD device may be delivered through segmented electrodes, active matrix thin film transistors, or digital micromirrors.

[0125] The term digital microfluidics (DMF) device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any device having only droplets in an oil stream in a channel. By activating specific electrodes, the droplets are moved across the surface by electrodynamic forces. Upon electrode activation, the dielectric layer becomes less hydrophobic, thus causing the droplets to spread across the surface. The setup of a digital microfluidics (DMF) device is known in the art and depends on the substrate used, the electrodes, the configuration of those electrodes, the use of dielectric material, the thickness of the dielectric material, the hydrophobic layer, and the applied voltage.

[0126] The electrokinetic device includes a first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating, the functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate including a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and a voltage source operably coupled to the matrix electrodes.

[0127] The dielectric layer may include 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 thickness of the dielectric layer may be 10 nm to 100 μm. A combination of more than one material may be used, and the dielectric layer may include more than one sublayer, which may be made of different materials.

[0128] The conformal layer may comprise parylene, siloxane or epoxy. It may be a thin protective parylene coating between an insulating dielectric and a hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In the present invention, the reason for depositing parylene is not to improve insulating / dielectric properties, such as reducing pinholes, but as a conformal layer between a dielectric layer and a hydrophobic layer. The inventors have found that, in contrast to other similar insulating coatings of the same thickness, such as PDMS (polydimethylsiloxane), parylene prevents contact angle hysteresis caused by high conductivity solutions or solutions that deviate from neutral pH for a long time. The thickness of the conformal layer may be from 10 nm to 100 μm.

[0129] The hydrophobic layer may comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite, a zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, a carbon nanotube structure, a silica nanocoating, or a slippery liquid-infused porous coating.

[0130] The element may include one or more of a plurality of array elements, each element containing element circuitry; discrete electrodes; a thin film semiconductor whose electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0131] The functional coating may comprise a dielectric layer comprising silicon nitride, a conformal layer comprising parylene and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0132] The electro-motion device may include a controller for regulating the voltage supplied to the individual matrix electrodes. The electro-motion device may include a plurality of scan lines and a plurality of gate lines, wherein each thin film transistor is coupled to a scan line and a gate line, and the plurality of gate lines are operably connected to the controller. This enables all individual elements to be individually controlled.

[0133] Device

[0134] Manipulation of droplets by applying an electric potential can be achieved on electrodes covered with an insulator or dielectric or a series of insulators or dielectrics. Manipulation of droplets by an applied electric potential is called electrowetting. The electromotion occurs due to an inhomogeneous electric field that affects the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of a liquid on a solid surface (electrowetting on dielectrics or EWoD). DEP can also be used to generate forces on polarizable particles to induce their movement. The electrical signal can be transmitted to discrete electrodes, transistors, transistor arrays or semiconductor membranes, whose electrical properties can be modulated by optical signals.

[0135] The EWoD phenomenon occurs when a droplet is actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in 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:

[0136]

[0137] in, is the contact angle when the electric field across the interfacial layer is zero, is the liquid-gas tension, c is the specific capacitance (as Given, where ε r is the dielectric constant of the insulator / dielectric, is the permittivity of a vacuum, t is the thickness), and V is the applied voltage or potential. Therefore, the change in contact angle (induced droplet movement) is a function of surface tension, potential, dielectric thickness, and dielectric constant.

[0138] When a droplet is actuated by an EWoD, there are two sets of opposing forces acting on it: the electrowetting force induced by the electric field and the drag force, which consists of the drag force caused by the interaction of the droplet with the filler dielectric and the contact line friction. The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all the drag forces is determined by the thickness of the insulator / dielectric and the dielectric contact ratio. Therefore, in order to reduce the actuation voltage, it is necessary to reduce (i.e. increasing the dielectric constant or decreasing the thickness of the insulator / dielectric). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, depositing 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 actuate the droplets. Therefore, most academic studies report using much higher voltages of >100V on easily fabricated thick dielectric films (>3 microns) to achieve electrowetting.

[0139] However, high voltage EWoD-based devices with thick dielectric films have limited industrial applicability, primarily due to their limited droplet multiplexing capabilities. The use of low voltage devices including thin film transistors (TFTs) and optically activated amorphous silicon layers (a-Si) has paved the way for industrial applications of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplexed manner. The driving voltage of TFTs or optically activated a-Si is low (typically <15V). The bottleneck in the fabrication of low voltage devices and, therefore, the adoption of 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.

[0140] Typically, electrodes (or array elements) for EWoDs are covered by either (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings include fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of such materials as hydrophobic coatings on dielectrics is generally <100 nm and may have defects in the form of pinholes or porous structures; therefore, it is particularly important that the insulator / dielectric is free of pinholes to avoid electrical shorts. Teflon TM 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 silicones, epoxies (such as SU-8), or parylenes (e.g., parylene N, parylene C, parylene D, or parylene HT). Teflon is a popular choice for hydrophobic insulators due to its minimal contact angle hysteresis and large contact angle with aqueous solutions. TM Parylene C is still used as a hydrophobic outer coating for these insulator / dielectric polymers. However, there are difficulties in reliably manufacturing pinhole-free coatings of parylene or SU-8 <1 μm; therefore, the thickness of these materials is generally kept at 2-5 μm at the expense of increased electrowetting voltage requirements. It has also been reported that conventional EWoD devices with parylene C are prone to breakage and instability for repeated droplet manipulation with cell culture media.

[0141] Multilayer insulator devices deposited with metal oxide and parylene C films have been used to create more robust insulators / dielectrics that can operate at lower applied voltages. Inorganic materials, such as metal oxides and semiconductor oxides, which are commonly used as “gate dielectrics” in the CMOS industry, have been used as insulators / dielectrics for EWoD devices. They offer the advantage of thin film deposition (<100 nm) using standard cleanroom methods. These materials are inherently hydrophilic, requiring additional hydrophobic coatings, and are susceptible to pinhole formation due to the thin film layer deposition process. Together with the need for lower voltage operation of EWoDs, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using insulators / dielectrics with high dielectric constants), and (2) optimizing fabrication methods to make the insulators / dielectrics pinhole-free to avoid dielectric breakdown.

[0142] The device can be used for any biochemical assay process involving high solute (ionic) strength solutions, where high concentrations of ions would otherwise degrade and hinder the use of prior art devices. The device is particularly advantageous for processes involving biomolecule synthesis, such as, for example, nucleic acid synthesis, for example using template-independent chain extension, or cell-free protein expression using a set of different nucleic acid templates.

[0143] An active matrix thin film transistor (AM-TFT) device is disclosed having a substrate with a plurality of electrodes, the device including a plurality of fluid inlet ports on at least two sides of the device, wherein the inlet ports on each side of the device are evenly spaced, and wherein the device is connected to a pump. The device may include two substrates, wherein at least one substrate has a plurality of electrodes, and the two substrates define parallel plates separated by a spacer to define a volume.

[0144] The device can be connected to a pump, such as a syringe pump, a peristaltic pump, a disc pump, a diaphragm pump or a pneumatic pump. The pump can fill the device with a filling fluid in an automated manner. Once filled, the pump can extract the filling fluid to create a negative pressure in the device, which draws the reagent from the hole. Thus, fluid extraction can be performed in an automated manner to achieve "hands-free" loading of aqueous reagents.

[0145] Application of the present invention

[0146] The present invention can be used in numerous different applications. In these applications, the steps of placing aqueous droplets having ionic strength on the first matrix electrode and providing a differential potential can be repeated many times. They can be repeated 1000 times or more than 10,000 times, sometimes repeated within a 24 hour period.

[0147] Application of Enzymatic DNA Synthesis

[0148] The method can be used to synthesize 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 3'-O-reversibly terminated nucleoside 5'-triphosphate addition to the 3'-end of a 5'-immobilized nucleic acid. During enzymatic nucleic acid synthesis, the following steps are taken on the instrument:

[0149] I. An addition solution containing TdT, optional pyrophosphatase (PPiase), 3'-O-reversibly terminated dNTPs and a desired buffer (comprising salts and necessary reaction components such as metal divalents) is brought to a reaction zone containing immobilized nucleic acids, wherein the nucleic acids are immobilized on a surface such as by magnetic beads via covalent attachment to the 5' end of the nucleic acids. The initially fixed nucleic acids may be referred to as starting oligonucleotides and may contain N nucleotides, such as 3-100 nucleotides, preferably 10-80 nucleotides, more preferably 20-65 nucleotides. The starting oligonucleotide may contain a cleavage site, such as a restriction site or a non-canonical DNA base, such as U or 8-oxoG. The addition solution may optionally contain a phosphate sensor, such as an E. coli phosphate binding protein conjugated to an MDCC fluorophore, to assess the quality of nucleic acid synthesis as a fluorescent output. dNTPs may be combined in proportion to prepare a DNA library, such as NNK synthesis.

[0150] II. Apply a wash solution in bulk or discrete droplets to the reaction zone to wash away the added solution. The wash solution typically has a high solute concentration (>1M NaCl).

[0151] III. Applying a deprotection solution in bulk 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.

[0152] IV. Applying a wash solution in bulk or discrete droplets to the reaction zone to wash away the deprotection solution.

[0153] V. Repeating steps I-IV until the desired sequence is synthesized, for example, repeating steps I-IV 10 times, 50 times, 100 times, 200 times, or 1000 times.

[0154] This method can be used to prepare oligonucleotide sequences via synthesis or assembly. The device allows synthesis and movement of limited sequences. Using this method, the starting sequence can be modified at a specific position above the electrode and an extended oligonucleotide can be prepared. Starting sequences at different positions can be exposed to different nucleotides, thereby synthesizing different sequences in different regions of the electrowetting device.

[0155] After synthesizing a defined set of different sequences in different regions of the electrowetting device, the sequences can be further assembled into longer continuous sequences by ligating two or more synthesized strands together.

[0156] Described herein is a method for preparing a continuous oligonucleotide sequence of at least 2n bases in length, comprising: using the electrowetting device described herein, the device having a plurality of fixed starting oligonucleotide sequences, one or more of which contain a cleavage site; using the starting oligonucleotide sequences to synthesize a plurality of fixed oligonucleotide sequences of at least n bases in length; using a cycle of extension of reversibly blocked nucleotide monomers; selectively cleaving at least two of the fixed oligonucleotide sequences of at least n bases in length into a reaction solution while keeping one or more fixed oligonucleotide sequences connected; hybridizing at least two of the cleaved oligonucleotides to each other to form a splint; hybridizing one end of the splint to one of the fixed oligonucleotide sequences, and connecting at least one of the cleaved oligonucleotides to the fixed oligonucleotide sequence, thereby preparing a continuous oligonucleotide sequence of at least 2n bases in length.

[0157] The present invention can be used to automate the movement of droplets in a cartridge. For example, droplets that are intended for analysis can be moved according to the present invention. The present invention can be incorporated into a cartridge for local clinician diagnosis. For example, it can be used in conjunction with nucleic acid amplification tests (NAATs) to determine nucleic acid targets in, for example, genetic testing for indications such as cancer biomarkers, pathogen testing, such as detecting bacteria in blood samples, or virus testing, such as coronaviruses, such as SARS-CoV-2 for COVID-19 diagnosis.

[0158] The device may be thermally cycled to achieve nucleic acid amplification, or the device may be maintained at a desired temperature for isothermal amplification.Having different synthesized sequences in different regions of the device enables multiplex amplification using different primers in different regions of the device.

[0159] Furthermore, the present invention can be used in conjunction with next generation sequencing, in which DNA is synthesized by adding nucleotides and a large number of samples are sequenced in parallel. The present invention can be used to accurately locate individual samples used in next generation sequencing.

[0160] The present invention can be used for the automation of library preparation for next generation sequencing. For example, the connection step of sequencing adapters can be performed on the device. Then, the amplification of a selected small group of sequences from the sample can have the connected adapters to achieve sequencing of the amplified group.

[0161] Protein expression applications

[0162] The method of moving aqueous droplets can also be used to help promote cell-free expression of peptides or proteins. Specifically, the method of the present invention can be used to move droplets in the electrokinetic device, the droplets containing nucleic acid templates and cell-free systems with components for expressing proteins in an oil-filled environment.

[0163] Disclosed herein is a method for real-time monitoring of in vitro protein synthesis, comprising:

[0164] a. in vitro transcription and translation of a protein of interest fused to a peptide tag; and

[0165] b. Monitoring the presence of the peptide tag using other polypeptides that produce a detectable signal in the presence of the peptide tag.

[0166] Disclosed herein is a method for monitoring cell-free protein synthesis in droplets on a digital microfluidics device, comprising:

[0167] a. Cell-free transcription and translation of a protein of interest fused to a peptide tag; and

[0168] b. Monitoring the presence of the peptide tag using other polypeptides that produce a detectable signal in the presence of the peptide tag.

[0169] The terms "in vitro" and "cell-free" are used interchangeably herein.

[0170] The detectable signal may be, for example, fluorescence or luminescence.The detectable signal may also result from the binding of the ligand to a complementary oligo-, peptide- or polypeptide tag fused to the protein of interest.

[0171] A detectable signal may also result from binding of the polypeptide to a protein of interest fused to a His-tag.

[0172] Any in vitro transcription and translation, such as extract-based systems derived from rabbit reticulocyte lysate, human lysate, Chinese hamster ovary lysate, wheat germ, HEK293 lysate, E. coli lysate, yeast lysate, can be used.

[0173] Alternatively, in vitro transcription and translation can be assembled from purified components, such as the Purified Recombinant Elements system (PURE).

[0174] In vitro transcription and translation can be coupled or uncoupled.

[0175] The peptide tag can be a component of a fluorescent protein, and the other polypeptide is a complementary part of the fluorescent protein. Fluorescent proteins can include sfGFP, GFP, ccGFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example, the peptide tag can be GFP 11 , other polypeptides can be GFP 1-10 The peptide tag may be a component of sfCherry. The peptide tag may be a component of sfCherry 11 , other polypeptides can be sfCherry 1-10 In the presence of a hydroxybenzylidene rhodanine analog, the peptide tag can be CFAST 11 or CFAST 10 , the other polypeptide may be NFAST.

[0176] The peptide tag may also be a component of a protein that forms a detectable substrate, such as a luminescent or chromogenic substrate. The protein may include β-galactosidase, β-lactamase or luciferase.

[0177] Proteins can be fused to multiple tags. For example, a protein can be fused to multiple GFP 11 Peptide tag fusion and multiple GFP 1-10 For example, a protein can be synthesized with multiple sfCherry 11 Peptide tag fusion and multiple sfCherry 11 The protein of interest can be synthesized with one or more sfCherry 11 Peptide tag and one or more GFP 11 Peptide tag fusion and one or more GFP 1-10 Peptide and one or more sfCherry 1-10 Synthesis occurs in the presence of polypeptides.

[0178] Any protein of interest can be synthesized. The protein can be an enzyme, such as terminal deoxynucleotidyl transferase (TdT) or a truncated form thereof, or a homologous amino acid sequence of terminal deoxynucleotidyl transferase (TdT) in other species, or a homologous amino acid sequence of Polμ, Polβ, Polλ and Polθ in any species, or a homologous amino acid sequence of X family polymerase in any species.

[0179] Protein expression generally requires an adequate oxygen supply. The most convenient and high-yield way to power CFPS is through oxidative phosphorylation, in which O 2serves as the final electron acceptor; however, there are other ways that involve supplementation with energy molecules that do not participate in oxidative phosphorylation. In confined droplet microfluidics or digital microfluidics systems, there is not enough oxygen available to achieve efficient protein synthesis.

[0180] Described herein are improved methods for achieving cell-free expression of peptides or proteins in digital microfluidic devices. Included is a method for cell-free expression of peptides or proteins in a microfluidic device, wherein the method includes one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and a cell-free system having components for expressing proteins in an oil-filled environment, and moving the droplets using electrowetting. The components for cell-free protein synthesis droplets can be pre-mixed before introduction into the digital microfluidic device, or mixed on the digital microfluidic device.

[0181] The droplets can be repeatedly moved for a period of at least 30 minutes while expressing the protein. The droplets can be repeatedly moved for a period of at least two hours while expressing the protein. The droplets can be repeatedly moved for a period of at least 12 hours while expressing the protein. The action of moving the droplets causes oxygen to be supplied to the droplets and dispersed throughout the droplets. The action of moving improves protein expression levels compared to droplets that remain stationary.

[0182] Any electrowetting method may be used to move the droplets. The droplets may be moved using electrowetting on dielectrics (EWoD). The electrical signals on an EWoD or optical EWoD device may be delivered through segmented electrodes, active matrix thin film transistors, or digital micromirrors.

[0183] The fill fluid in the device can be any water-immiscible liquid. The fill fluid can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), alkyl solvents such as decane or dodecane, or fluorinated oils. The fill fluid can be oxygenated before or during the expression process.

[0184] A source of supplemental oxygen can be supplied to the droplets. For example, during protein expression, droplets or bubbles containing gaseous or dissolved oxygen can merge with the droplets. In addition, the source of supplemental oxygen can be obtained by oxygenating the oil used as a filler medium. It is well known in the art that oils, such as hexadecane, HFE-7500, etc. can be oxygenated to support cell growth, particularly the oxygen demand of E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.

[0185] Droplets can be formed prior to entering a microfluidic device and flow into the device. Alternatively, droplets can be merged on the device. A method is included that includes merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free extract having components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0186] The droplet can be split on the device before or after expression. Included herein is a method further comprising splitting the aqueous droplet into a plurality of droplets. If desired, the split droplets can be screened with other additives. Included is a method wherein one or more split droplets are combined with an additive droplet for screening.

[0187] The cell-free expression of peptide or protein can use cell lysate with the reagent realizing protein expression.The common components of cell-free reaction include energy source, amino acid supply, auxiliary factor such as magnesium, and relevant enzyme.Cell extract is obtained by cracking the cell of interest and removing cell wall, DNA genome and other fragments by centrifugation.The rest are cell machinery, including ribosome, aminoacyl-tRNA synthetase, translation initiation factor and elongation factor, nuclease etc.Once add suitable nucleic acid template, the expression machinery of cell origin can be used to express nucleic acid template as peptide or protein.

[0188] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used for CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates that can be produced in cells or produced synthetically. LETs can be prepared via PCR. Although LETs are easier and faster to prepare, the yield of plasmids is generally higher in CFPS. mRNA can be produced by an in vitro transcription system. The method uses a single nucleic acid template per droplet. The method can use multiple droplets with different nucleic acid templates per droplet.

[0189] The energy source is an important part of the cell-free reaction. Typically, a separate mixture containing the required energy source is added to the extract for the reaction along with a supply of amino acids. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. During expression, the energy source can be supplemented by adding other reagents to the droplets during the process.

[0190] The cell-free extract with components for protein expression contains everything required for protein expression except the nucleic acid template. Therefore, the term includes all related ribosomes, enzymes, initiators, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, the expression of the protein begins without the need for other reagents.

[0191] Therefore, the cell lysate may be supplemented with additional reagents before the template is added. Cell-free extracts with components for protein expression will generally be made as a bulk reagent or 'master mix', which can be formulated into many identical droplets before different templates are added to different droplets separately. Common cell extracts currently in use are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Kluyveromyces yeast (D2P system). All of these extracts are commercially available.

[0192] Cell-free systems can be assembled from the required reagents, not from cell extracts. Systems based on reconstructed, purified molecular reagents are commercially available, such as the PURE system for making proteins, and can be used as provided. The PURE system consists of all enzymes involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reactions of the PURE system lack proteases and ribonucleases, which are often present as undesirable molecules in cell extracts.

[0193] Once the CFPS reagents have been enclosed in a droplet, additional reagents can be provided by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including, for example, an oxygen or 'power' source, or a test agent that is desired to be exposed to the expressed protein.

[0194] The droplets may be aqueous droplets. The droplets may contain an oil-immiscible organic solvent, such as, for example, DMSO. The droplets may be a mixture of water and a solvent, provided that the droplets do not dissolve into the bulk filler.

[0195] Thus, droplets containing a cell-free extract with components for protein expression will generally be in an oil-filled environment before the nucleic acid template is added to the droplets. The template can be added by merging droplets on the microfluidic device. Alternatively, the template can be added to droplets outside the device and then flowed into the device for the expression process. For example, the expression process can be initiated on the device by increasing the temperature. Expression systems generally operate best at temperatures above standard room temperature, such as at or above 29°C.

[0196] The expression process generally takes several hours. Therefore, the process should be maintained for at least 30 minutes or 1 hour, generally at least 2 hours. Expression can be maintained for at least 12 hours. During the expression process, the droplets should be moved within the device. Movement can improve the process by mixing the reagents and ensuring that there is enough oxygen available within the droplets. Movement can be continuous, or it can be repeated with intermediate time periods without movement.

[0197] Thus, while expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least 30 minutes or 1 hour. While expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least two hours. While expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least 12 hours. The action of moving the droplets enables mixing within the droplets and enables the supply of oxygen or other agents to the droplets. The action of moving improves protein expression levels compared to droplets that remain stationary.

[0198] Through affinity tags, such as FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag or other forms of affinity tags, CFPS expressed proteins can be immobilized on solid support affinity resins, and fresh batches of CFPS reagents can be delivered on the resin. Therefore, updated reagents can be used to perform protein synthesis, closely mimicking the industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS manufacturing methods.

[0199] The droplets may also contain additives to reduce the effects of biofouling on the digital microfluidic surface. Specifically, the droplets containing the CFPS component may also contain additives, such as surfactants or detergents, to reduce the effects of biofouling on the hydrophobic or superhydrophobic surfaces of the 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. Specifically, the droplets containing the CFPS component may contain 0.1% volume / volume TWEEN20, 0.1% volume / volume Triton X-100, and / or 0.08% weight / volume Pluronic F127.

[0200] Instead of adding a surfactant to the aqueous sample, a surfactant, such as a sorbitan ester, such as Span85 (e.g., Sorbitan trioleate, Sigma Aldrich, SKU8401240025), can be added to the fill solution. This has the advantage of allowing the CFPS reaction to be performed in DMF without dilution or doping. In addition, it simplifies the sample preparation process for setting up the reaction, improving ease of use and consistency of results. Using 1% weight / weight Span85 in dodecane enables undiluted CFPS reactions in DMF and undiluted detection of expressed non-fluorescent proteins. Surfactants other than Span85 and oils other than dodecane can be used. A range of concentrations of Span85 can be used. The surfactant can be nonionic, anionic, cationic, amphoteric, or mixtures thereof. The oil can be a mineral oil or a synthetic oil, including silicone oil, petroleum oil, and perfluorinated oils. Surfactants can have adverse effects on (1) the CFPS reaction and (2) the efficiency of the detection system (if the detection system involves complementation of the label and the analyte). For example, by using an oil-surfactant mixture for the CFPS reaction on DMF, detection of the expressed protein can also be performed without dilution and without the addition of aqueous surfactant. Surfactants have been shown to reduce the expression of some detection systems, including but not limited to isolated GFP (e.g., GFP 11 / ccGFP 1-10 ) system, it would be beneficial to remove the surfactant from the reagent mixture and instead add it to the oil.

[0201] The peptide tag can be attached to the C-terminus or N-terminus of the protein. The peptide tag can be a component of green fluorescent protein (GFP). For example, the peptide tag can be GFP 11 , the other polypeptide can be GFP 1-10 The peptide tag may be a component of sfCherry. The peptide tag may be a component of sfCherry 11 , and the other polypeptide can be sfCherry 1-10 .

[0202] A protein can be fused to multiple tags. For example, a protein can be fused to multiple GFP 11 Peptide tag fusion and multiple GFP 1-10 For example, a protein can be synthesized with multiple sfCherry 11 Peptide tag fusion and multiple sfCherry 1-10 The protein of interest can be synthesized with one or more sfCherry 11 Peptide tag and one or more GFP 11Peptide tag fusion and one or more GFP 1-10 Peptide and one or more sfCherry 1-10 Synthesis occurs in the presence of polypeptides.

[0203] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components (with or without the other). For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were listed individually herein. Unless the context dictates otherwise, the descriptions and definitions of the above features are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0204] It will be further 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 may be constructed without departing from the scope of the invention as defined in the appended claims.

[0205] Example loading scenario:

[0206] Step 1 – Filling the Digital Microfluidics Device with Water-Immiscible Filler Fluid

[0207] 1.5 mL of dodecamethylpentasiloxane (DMPS) containing 0.1% volume / volume Span85 (filling fluid) was introduced into the digital microfluidic device via an automated syringe pump (Chemyx Fusion 200X) at a flow rate of 1 mL / min. After a waiting period of 30 seconds, the filling fluid was allowed to reach the level in all loading wells, and then another 0.5 mL of filling fluid was introduced at 0.5 mL / min. This resulted in the digital microfluidic device and all loading wells being filled with the filling fluid.

[0208] Step 2 – Transfer aqueous reagents into the loading port

[0209] Use an 8-channel multichannel pipette to load 3-12 μL of aqueous reagent into different wells. Depending on the desired reservoir, load 3 μL in some ports, 5 μL in some ports, and 12 μL in some ports.

[0210] Step 3 – Pumping out fill fluid to facilitate entry of aqueous reagents into the digital microfluidics device

[0211] The same syringe pump was used to draw 0.7 mL of filling fluid from the digital microfluidic device at a flow rate of 0.1 mL / min. At the end of this step, the aqueous reagent had entered the digital microfluidic device, made contact with the electrode substrate, and thus formed a reservoir through electrode activation. The loading process can be seen in Figure 7 。

Claims

1. A method for loading a plurality of volumes of aqueous liquids of different sizes into a digital microfluidic device having a substrate with a plurality of electrodes, the method comprising: a. providing a device having a plurality of holes, wherein the plurality of holes are connected to a substrate having a plurality of electrodes via an inlet; b. filling the device with a water-immiscible filling liquid, such that the plurality of holes are at least partially filled with the filling liquid; c. loading two or more of the wells with an aqueous liquid dropped into the wells; as well as d. A portion of the filling liquid is withdrawn from the device, thereby drawing the aqueous liquid onto the plurality of electrodes through the inlet to form a plurality of volumes of aqueous liquid of different sizes.

2. The method of claim 1, wherein: The digital microfluidics device is an active matrix thin film transistor (AM-TFT) based device.

3. The method according to claim 1 or claim 2, wherein: At least eight reservoirs of aqueous liquid are formed simultaneously on the device.

4. The method according to any one of claims 1 to 3, wherein: The device is loaded simultaneously from two or more sides of the device, with a different volume being loaded from each side.

5. The method according to any one of claims 1 to 4, wherein The digital microfluidics device includes two parallel plates separated by a spacer to define a fluid volume.

6. The method according to any one of claims 1 to 5, wherein The filling liquid is a hydrophobic liquid or a non-ionic liquid.

7. The method according to any one of claims 1 to 6, wherein The inlet is in the top substrate and the plurality of electrodes are on the bottom substrate.

8. The method of claim 5, wherein the spacer comprises an adhesive having beads of defined size distribution, a glass of defined thickness, or a polymer material.

9. The method according to any one of claims 5 to 8, wherein The spacers define a gap of 50 microns to 250 microns.

10. The method according to any one of claims 1 to 9, wherein The filling fluid is aspirated from the device by a syringe pump, peristaltic pump, disc pump, diaphragm pump or pneumatic pump.

11. The method according to any one of claims 1 to 10, wherein The aqueous liquid is introduced into the wells by a pipette, multichannel pipette, syringe, blister pack, acoustic dispenser, or robotic liquid handler.

12. The method of any one of claims 1 to 11, wherein Once the aqueous liquid has been adsorbed onto the substrate with the plurality of electrodes, a small group of the plurality of electrodes is actuated to control the position of the aqueous liquid.

13. The method of any one of claims 1 to 12, wherein The inlet has a diameter of 0.2 mm to 2 mm, for example, a diameter of 1 mm.

14. The method of any one of claims 1 to 13, wherein Each well has a single inlet.

15. The method of any one of claims 1 to 14, wherein The filling is performed at 0.01 to 2 mL / min.

16. The method of any one of claims 1 to 15, wherein The removal was performed at 0.01 mL / min to 2 mL / min.

17. The method of any one of claims 1 to 16, wherein An aqueous fluid introduced into the device forms an aqueous reservoir and includes temporarily actuating electrodes on the side of the reservoir opposite the source fluid to form one or more visual calibration structures, which are the last areas to fill and once filled indicate that a precise volume of aqueous fluid has been introduced, so that when the temporarily actuated electrodes are closed, the liquid becomes part of the reservoir, thereby precisely controlling the liquid area in the reservoir.

18. The method of any one of claims 1 to 17, wherein The reservoir comprises X by Y pixels, where X is the width in the range of 10-50 pixels and Y is the length in the range of 10-150 pixels.

19. A method according to any preceding claim, wherein: The aqueous reagents loaded into the device contain DNA constructs and / or reagents for cell-free protein synthesis.

20. The method according to any one of claims 1 to 19, wherein: The filling liquid is dodecamethylpentasiloxane, decane or dodecane.

21. The method according to any one of claims 1 to 20, wherein: The filling liquid contains a surfactant.

22. The method according to claim 21, wherein: The surfactant is sorbitan ester.

23. A method according to any preceding claim, wherein: At least 32 volumes of aqueous liquid are formed, the aqueous liquid having at least 2 different sizes.

24. A method according to any preceding claim, wherein: The sides of the hole are shaped to form a continuous curve without inflection points.

25. A method according to any preceding claim, wherein: An automated syringe pump was used to withdraw the filling solution through two ports at different corners of the device.

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