Droplet interfaces in electro-wetting devices

Abstract

A droplet interface is formed between droplets within an electro-wetting device comprising an array of driving electrodes. A driving signal is applied to selected driving electrodes to bring two droplets into close proximity, placing the droplets in an energized state where the shape of the droplets is deformed compared to the shape of the droplets in a lower energy state. Subsequently, the driving signal is changed to lower the energy of the droplets to a lower energy state, causing the droplets to relax into a gap, and the two droplets come into contact with each other to form a droplet interface. The use of a sensing electrode in the device enables the measurement of electric current across the droplet interface. The sensing electrode can be used to (i) apply a reference signal during droplet driving, or (ii) record electric current measurements.

Description

Technology Field

[0001] In some embodiments, the present invention relates to an electro-wetting element and to a use thereof for forming a droplet interface between droplets in a fluid medium. In some embodiments, the present invention relates to an electro-wetting element, the use thereof for taking measurements on a droplet interface formed by electro-wetting.

[0002] Electro-wetting devices, such as electro-wetting on dielectric (EWOD) devices, are known to manipulate droplets in a fluid medium.

[0003] To examine this in more detail, dielectric electrowetting is a well-known technique for manipulating fluid droplets by applying an electric field, as disclosed, for example, in U.S. Patent Application Publication US2016 / 0305906. Examples of the configuration and operation of EWOD devices are described in the following literature. U.S. Patent US-6,911,132 discloses a two-dimensional EWOD array for controlling the position and movement of droplets in two dimensions. U.S. Patent US-6,565,727 discloses other droplet manipulation methods, including splitting and merging droplets and mixing droplets of different materials together. U.S. Patent US-7,163,612 describes a method for using an active matrix (AM) arrangement based on thin-film electronic devices including thin-film transistors (TFTs) to control the addressing of voltage pulses for EWOD devices, using a circuit arrangement similar to those employed in AM display technology. Such a general type of device may be referred to as an AM-EWOD device.

[0004] It has been proposed to manipulate these droplets using an EWOD device to form droplet interfaces between the droplets, for example, comprising a membrane of amphiphilic molecules. This provides a potentially useful system for studying the droplet interface itself and also provides processes occurring at the droplet interface. In one example of particular interest, these processes may include inserting a membrane-penetrating pore and subsequently measuring characteristics such as ion current flow, which may vary depending on the interaction between this membrane-penetrating pore and the analyte.

[0005] Membranes of amphiphilic molecules, such as artificial planar lipid bilayers, can serve as simplified models of biomembranes and are widely used in the study of various processes, including the characterization of transmembrane pores, such as transmembrane protein pores and ion channels. Ion channels are a diverse group of transmembrane protein pores that selectively control the movement of specific ions across cell membranes in biology, thereby establishing fundamental voltage and electrochemical gradients for a wide variety of biological processes.

[0006] Single-channel recording (SCR) of individual protein vacancies is a powerful tool for studying channel protein function. Single-channel recording can measure changes in ion-current through a single protein channel and examine voltage dependence, gating behavior, ligand binding affinity, and ion selectivity at the single-molecule level. For example, various methods can be used to form lipid bilayers as disclosed in the literature [Montal, M. & Mueller, p. 1972 Proceedings of the National Academy of Sciences of the United States of America 69, 3561-3566]. Although planar lipid bilayers are widely used, they are difficult to manufacture, and their short lifetime hinders their application in many situations.

[0007] Accordingly, other membranes of amphiphilic molecules have been proposed. Alternatives to planar lipid bilayers have been disclosed; for example, International Publication WO-2008 / 012552, which discloses a method for forming a bilayer of amphiphilic molecules, uses droplets of an aqueous solution in a hydrophobic medium, such as oil.

[0008] An array of individual suspended membranes of amphiphilic molecules containing respective ion channel protein pores or nanopores may be provided, as disclosed, for example, in International Publication WO-2014 / 064443. The ion current between two aqueous solutions provided on either side of the amphiphilic membrane may be measured to characterize an analyte, e.g., a polynucleotide, and a MinION comprising a nanopore array capable of determining the polynucleotide sequence may be provided. TM Commercial devices such as the above are sold by Oxford Nanopore Technologies Ltd.

[0009] Therefore, the droplet interface between droplets in contact with each other is an alternative method for forming a membrane of amphiphilic molecules. Such a membrane formed by a bilayer of amphiphilic molecules can be referred to as a droplet interface bilayer (DIB). Multiple membranes can be formed at the interface between multiple droplet pairs. Techniques for forming a DIB are disclosed, for example, in the literature [Leptihn et al, Nature Protocols 8, 1048-1057 (2013)], and the DIB can be used to study the membrane-penetrating pores inserted into the DIB. For example, the literature [Martel et al., Biomicrofluidics 6, 012813 (2012)] discloses a microfluidic device for forming a droplet interface bilayer with inserted protein channels. Gold microwires were deposited on a substrate containing actuation electrodes provided with Ag / AgCl pads to electrically contact each droplet in order to perform measurements of ion current flow through the membrane channel.

[0010] A first aspect of the present invention relates to a method and an apparatus for forming a droplet interface in an electro-wetting device.

[0011] According to a first aspect of the present invention, a method for forming a droplet interface in an electro-wetting device is provided, wherein the electro-wetting device comprises: an array of driving electrodes; and an insulating layer covering the driving electrodes and having an outermost hydrophobic surface; Two droplets comprising a fluid medium and a liquid within the fluid medium are disposed on the hydrophobic surface, wherein one of the liquid and the fluid medium is polar and the other of the liquid and the fluid medium is nonpolar, thereby, the driving electrodes can electro-wet the droplets when a driving signal is applied to the driving electrodes, and the method comprises: applying a driving signal to selected driving electrodes to place one or both of the two droplets in an energized state in which the shape of the one or both droplets is deformed compared to a lower energy state, and bringing the two droplets close together with a gap between them, wherein the gap is selected such that the two droplets do not come into contact with each other when one or both of the droplets are in the energized state, and the two droplets come into contact with each other when they are in the lower energy state to form a droplet interface; and to lower the energy of one or both of the droplets, the driving signal applied to the driving electrodes is changed to a lower energy state, thereby causing one or both of the droplets to relax the gap and form a droplet interface by the two droplets coming into contact with each other.

[0012] This method is applied to an electro-wetting device comprising an array of driving electrodes and an insulating layer covering the driving electrodes and having an outermost hydrophobic surface. In such an electro-wetting device, two droplets comprising a fluid medium and a liquid in the fluid medium may be placed on the hydrophobic surface. The driving electrodes electro-wet the droplets when a driving signal is applied thereto, thereby enabling manipulation of the droplets by the selection of the driving signal.

[0013] The above method provides a reliable technique for forming a droplet interface between two droplets. In the method of the present invention, a driving signal is applied to driving electrodes in two phases. In the initial phase, the applied driving signal has a predetermined pattern selected to place one or both of the two droplets in an energy-gaining state. As a result, the shape of one or both droplets is deformed compared to when they are in a lower energy state. In this energy-gaining state, the two droplets come close together with a gap between them. The gap is selected so that the two droplets do not come into contact with each other when one or both of the two droplets are in the energy-gaining state, but come into contact with each other when they are in the lower energy state to form a droplet interface.

[0014] In a subsequent step, the applied driving signal is changed to lower the energy of one or both of the droplets to a lower energy state. As a result, one or both droplets relax into the gap, and the two droplets come into contact with each other. Thus, a droplet interface is formed between the two droplets. Therefore, the movement of the surface of one or both droplets is caused by the relaxation of the energy-gained state, which is a passive process.

[0015] This process improves the reliability of droplet interface formation compared to attempts to bring two droplets into direct contact by applying a driving signal that moves the entire droplet toward each other. Although this method is possible in principle, droplets tend to fuse (i.e. integrate), making it difficult to maintain an interface between droplets.

[0016] Although the above method can be applied by placing two droplets into an energy-gaining state one by one, preferably both droplets are placed into an energy-gaining state. As a result, the surfaces of both droplets relax into the gap and come into contact with each other to form a droplet interface. In this way, the relaxation of both droplets is used to form a droplet interface, which further increases the reliability of the formation.

[0017] In the energy-gaining state of one or both droplets, the shape of the droplets can be selected such that the desired relaxation of the surface allows the droplet interface to be formed. Although various shapes are possible, advantageously, the shape of the droplets in the energy-gaining state is elongated when viewed from the plane of the electro-wetting device. Similarly, the shape of the contact line of the droplets in the energy-gaining state is elongated. In this case, the gap between the droplets can be extended along the long axis of the elongated shape so that, upon relaxation, the surface of the droplet extending along the long axis can move into the gap and come into contact with another droplet.

[0018] When an elongated shape is used, the shape of the droplet in the energy-gaining state may have an aspect ratio of at least 2:1, preferably at least 4:1 or at least 8:1. Generally, increasing the aspect ratio increases the degree of movement of the surface of at least one droplet, which helps the droplets come into contact.

[0019] During the step of applying a driving signal to the driving electrodes, two droplets may come close to the extent that the centroids of the two droplets are separated by a distance shorter than the combined radius of the droplets along the line between the centroids of the droplets in the lower energy state of the droplets.

[0020] The above method can be advantageously applied to an electro-wetting device in which the region surrounded by the contact line of a droplet in a lower energy state covers at least two driving electrodes, preferably at least five driving electrodes, at least ten driving electrodes, or at least twenty driving electrodes. Generally, in the design of an electro-wetting device, the more driving electrodes covered by the droplet, the better the resolution of shape control in the energy gain state of the droplet becomes. In other words, this can increase the degree of movement of the surface of at least one droplet, which helps the droplet contact.

[0021] A driving signal selected to impart energy to one or more droplets may be an alternating current (AC) driving voltage signal. Generally, the use of an AC driving signal in an electro-wetting device is known to be advantageous for manipulating droplets. In this case, preferably, the step of changing the driving signal applied to the selected driving electrodes may include the step of applying a DC potential or a floating potential to the selected driving electrodes instead of the AC driving signal. This improves the reliability of droplet interface formation because the DC potential or floating potential is less likely to rupture the droplet interface than when the AC driving signal is maintained.

[0022] The above method can be applied to an electro-wetting device comprising a layer of electrical insulating material coated with a hydrophobic material that forms the hydrophobic surface, wherein the insulating layer is an insulating layer.

[0023] The above method may be applied to an electro-wetting device further comprising a second substrate facing a hydrophobic surface of an insulator layer, wherein the second substrate is coated with a hydrophobic material forming an additional hydrophobic surface facing the hydrophobic surface of the insulator layer. In this case, a droplet may be placed on the hydrophobic surface of the insulator layer as well as on the additional hydrophobic surface of the hydrophobic layer. In this way, the droplet is sandwiched between the two substrates, which constrains the shape of the droplet. This improves the degree of control over the shape of the droplet between an energy-gain state and a lower energy state, which in turn improves the reliability of the formation of the droplet interface.

[0024] Furthermore, the second substrate can support sensor electrodes that form an electrical connection with the droplets formed between the droplet interfaces.

[0025] The above method can be applied to an electro-wetting device further comprising an active matrix array connected to driving electrodes.

[0026] The above method may be applied to form only a single droplet interface between two droplets, but likewise the method may be applied with one or more additional droplets placed on a hydrophobic surface, and a step of applying and changing a driving signal to driving electrodes may be performed to form multiple droplet interfaces between multiple pairs of droplets.

[0027] After the formation of the droplet interface, electrical measurements can be performed between the droplets across the droplet interface. For example, the electrical measurements may be measurements of ion flow through the membrane-penetrating pores between the droplets, or may be performed while applying a potential difference between the droplets.

[0028] Furthermore, according to a first aspect of the present invention, an electro-wetting element for forming a droplet interface that performs a method similar to that described above is provided.

[0029] A second aspect of the present invention relates to forming an electrical connection for each droplet in a system of droplets formed in an electro-wetting element and having one or more droplet interfaces between the droplets. Such electrical connection may be intended for the purpose of detecting droplet characteristics, such as the size or location of the droplets, when performing various droplet operations in the electro-wetting element, or for the purpose of performing measurements across the droplet interfaces.

[0030] U.S. Patent Application Publication US-2010 / 0,194,408 discloses, among other things, a method, circuit, and apparatus for detecting capacitance on a droplet driver for determining the presence, partial presence, or absence of a droplet at driving electrodes. U.S. Patent US-8,653,832 describes a method in which an impedance (or capacitance) sensing function can be integrated into the array element circuit of each array element of an AM-EWOD device, wherein the impedance sensor circuit can be used to determine the presence and size of a droplet present at each electrode in the array. However, these approaches are limited by the need to obtain a signal from the same electrode to which the driving signal is applied.

[0031] The literature [Martel et al., Biomicrofluidics 6, 012813 (2012)] discloses a microfluidic device for forming a droplet interface bilayer with an inserted protein channel, wherein gold microwires are deposited on a substrate containing driving electrodes, and Ag / AgCl pads on the electrodes are provided to make electrical contact with each droplet to perform measurements of ion currents flowing through the membrane channels. However, this construction is not only inconvenient and difficult to manufacture, but also limits the reliability of performing measurements and limits the scalability of the technique.

[0032] According to a second aspect of the present invention, an electro-wetting element for performing measurements across a droplet interface is provided, wherein the electro-wetting element comprises: a first substrate supporting an array of driving electrodes; an insulating layer covering the driving electrodes and having a hydrophobic surface; a second substrate facing the hydrophobic surface of the insulating layer and supporting at least one set of at least two sensor electrodes, wherein the electro-wetting element is configured to receive a droplet comprising a fluid medium and a liquid in the fluid medium disposed on the hydrophobic surface, and the driving electrodes are configured to receive a driving signal for electro-wetting to form at least one system of droplets having one or more droplet interfaces between the droplets, and each set of sensor electrodes is configured to make an electrical connection to each droplet in the at least one system of droplets.

[0033] Accordingly, in the present electro-wetting device, a sensor electrode is provided on a second substrate facing a hydrophobic surface of an insulating layer covering driving electrodes. This sensor electrode is arranged in at least one set of at least two sensor electrodes, and each set of sensor electrodes is configured to make an electrical connection to each droplet in a droplet system. This provides a convenient and reliable method of making an electrical connection to a droplet.

[0034] The second substrate may be coated with a hydrophobic material that forms an additional hydrophobic surface opposite to the hydrophobic surface of the insulator layer. In this case, the electro-wetting device may be configured to accommodate a fluid medium and a droplet placed on the additional hydrophobic surface of the hydrophobic layer as well as on the hydrophobic surface of the insulator layer. In this way, the droplet is sandwiched between the two substrates, which constrains the shape of the droplet. This improves the degree of control of the droplet by the driving signal applied to the driving electrodes.

[0035] When the second substrate is coated with a hydrophobic material, the hydrophobic material coating the second substrate may have an opening that exposes at least a portion of the sensor electrode. This improves the electrical connection between the sensor electrode and the droplets.

[0036] The second substrate additionally supports at least one additional electrode for receiving, for example, a reference signal while a driving signal is applied to the driving electrodes to manipulate the droplet.

[0037] If provided, the sensor electrode and additional electrode may be deposited on the surface of a second substrate facing the first substrate. In this case, if provided, the additional electrode may extend around the sensor electrode.

[0038] The electro-wetting element may further include a control system connected to driving electrodes, configured to apply a driving signal to the driving electrodes to manipulate a received droplet.

[0039] The control system may be configured to apply a reference signal to the additional electrode if the sensor electrode and / or additional electrode is provided while applying a driving signal to the driving electrodes.

[0040] The control system may be configured to apply a driving signal to driving electrodes selected to form a droplet interface at the interface between two droplets.

[0041] The electro-wetting element may further include a sensor system connected to sensor electrodes, said sensor electrodes electrically connected to each droplet forming a droplet interface between them, said sensor system configured to perform electrical measurements, for example, including impedance measurements. Such electrical measurements may be performed across the droplet interface between two droplets in the droplet system.

[0042] The sensor system may be configured to perform electrical measurements between each sensor electrode making contact between each droplet across a droplet interface comprising a membrane of amphiphilic molecules having a membrane-penetrating pore inserted within the membrane, for example, measurements of ion flow through the membrane-penetrating pore between droplets and / or electrical measurements depending on the analyte interacting with the membrane-penetrating pore.

[0043] The sensor system may additionally include an analysis system configured to process electrical measurements to analyze the analyte interacting with the membrane-penetrating pores. For example, if the analyte is a polymer containing polymer units, the analysis system may be configured to process electrical measurements to derive the estimated identity of the polymer units of the polymer.

[0044] A third aspect of the present invention relates to the use of an electro-wetting device for performing experiments on a droplet interface.

[0045] According to a third aspect of the present invention, an apparatus for performing an experiment on a droplet interface is provided, the apparatus comprising an electro-wetting element comprising an array of driving electrodes and an insulating layer covering the driving electrodes and having an outermost hydrophobic surface, wherein the electro-wetting element is configured to receive a droplet comprising a fluid medium and a liquid in the fluid medium disposed on the hydrophobic surface; a control system configured to apply a driving signal to driving electrodes selected to manipulate the received droplet and form at least one system of droplets having one or more droplet interfaces between the droplets; and a sensor system configured to perform an electrical measurement between droplets in the system formed across the droplet interface.

[0046] This device is suitable for conducting experiments on droplet interfaces.

[0047] The above device includes an electro-wetting element in which a droplet interface can be formed. The electro-wetting element includes an array of driving electrodes and an insulating layer covering the driving electrodes and having an outermost hydrophobic surface. The electro-wetting element can receive a droplet comprising a fluid medium and a liquid in the fluid medium disposed on the hydrophobic surface.

[0048] The above device further includes a control system configured to apply a driving signal to the driving electrodes selected to manipulate the received droplets and form at least one system of droplets having one or more droplet interfaces between the droplets. Thus, the use of the control system enables the formation of droplet interfaces in the electro-wetting device.

[0049] The above device further includes a sensor system configured to perform electrical measurements across the droplet interface between droplets within the formed system, thereby enabling experiments to be performed on the formed droplet interface.

[0050] After the formation of the droplet interface, various types of electrical measurements can be performed across the droplet interface between the droplets. For example, the electrical measurement may be a measurement of ion flow through the membrane-penetrating pores between the droplets, or may be performed while applying a potential difference between the droplets.

[0051] The sensor system may further include an analysis system configured to process electrical measurements to analyze the analyte interacting with the membrane-penetrating pores. For example, if the analyte is a polymer containing polymer units, the analysis system may be configured to process electrical measurements to derive the estimated identity of the polymer units of the polymer.

[0052] Advantageously, the control system may be configured to modify at least one formed droplet system of droplets in response to an electrical measurement performed by a sensor system. The ability of the device to modify the formed droplet system of droplets based on feedback from the sensor system provides a significant advantage, as it enables the device to perform experiments on droplet interfaces in an adaptive manner.

[0053] The output of the sensor system to which the control system responds may include the electrical measurement itself. This provides a first type of control for experiments performed based on the electrical characteristics being measured. Since electrical characteristics are fundamental to related processes, such as the formation of droplet interfaces and reactions occurring at those interfaces, this first type of control can allow these processes to be taken into account and adaptedly modified.

[0054] Alternatively, if the sensor system includes an analysis system configured to process electrical measurements, the output of the sensor system includes the output of the analysis system. This provides a second type of control for experiments performed based on the analysis. Since this analysis enables obtaining higher-level information, for example, information regarding the analyte being analyzed, this second type of control provides powerful experimental adaptation based on the results of the analysis.

[0055] Since the types of control that can be performed are extensive, powerful experimental setups can be provided. Some non-limiting examples are as follows.

[0056] The formed droplet system can be modified by separating the droplet interfaces within the system.

[0057] The formed droplet system can be modified by moving a new droplet in the droplet system to come into contact with the current droplet and forming a droplet interface between the new droplet and the current droplet.

[0058] The formed droplet system can be modified by moving a new droplet into contact with a current droplet within the droplet system and fusing the new droplet with the current droplet. In this case, it may be possible for the new droplet not to contain amphiphilic molecules at the interface between the liquid and fluid medium of the droplet.

[0059] Advantageously, the control system may be configured to apply a driving signal to driving electrodes selected to form multiple droplet systems simultaneously. This allows the device to perform experiments on multiple droplet systems in parallel, thereby increasing the experimental throughput of the device.

[0060] The device may further include a droplet generation system configured to form a droplet disposed on the hydrophobic surface of an electro-wetting element in a fluid medium. In this case, a control system may be configured to control the droplet generation system to form the droplet. This increases the experimental capability of the device, as it allows the device to form a droplet containing a suitable reagent for experimental purposes.

[0061] The first to third embodiments of the present invention may be practiced together, for example, in the same device or apparatus. Therefore, preferred features of the first to third embodiments of the present invention, some of which are defined in the following dependent claims, may be combined in any combination. Brief explanation of the drawing

[0062] For better understanding, embodiments of the present invention will now be described by non-limiting examples with reference to the accompanying drawings, in which: Figure 1 is a schematic diagram of a device including an AM-EWOD element. Figure 2 is a schematic perspective view of the AM-EWOD device. Figure 3 is a cross-sectional view of a part of the AM-EWOD device. Figures 4a and 4b are diagrams of the circuit of the electrical load present on the driving electrode in the presence and absence of a liquid droplet, respectively. Figure 5 is a plan view of a thin-film electronic device in an AM-EWOD device. Figure 6 is a diagram of the array element circuit of the AM-EWOD device. FIG. 7 is a plan view of a layer of conductive material formed on a second substrate of an AM-EWOD device. FIGS. 8 to 12 are plan views of a pair of droplets on an array of driving electrodes in an AM-EWOD device during a successive step of a method for forming a droplet interface between droplets. FIGS. 13 and FIGS. 14 are plan views of each method forming two systems of three droplets. Figures 15 and 16 are plan views of each droplet system. Figure 17 shows two images of an array of driving electrodes in an AM-EWOD device. Figure 18 is a diagram of a patterned layer of conductive material in an AM-EWOD device. Figure 19 is an image of an array of driving electrodes in an AM-EWOD device in which three systems of two droplets are formed. Figure 20 is an image of a set of two droplets, each consisting of three droplets, before and after two droplet interfaces are formed between the droplets. Figure 21 is a diagram of electrical measurements of current and voltage during the formation, separation, and re-formation of the droplet interface in the AM-EWOD device. Figure 22 is an image of a system of two droplets in an AM-EWOD device. Figure 23 is a trace of the current signal obtained from the droplet system of Figure 22. Figure 24 is a graph of electrical measurements of current and voltage during an experiment in which DNA is translocated through the pores in a two-droplet system in an AM-EWOD device. Figure 25 is a diagram of the electrical measurements of current for six electric events. Specific details for implementing the invention

[0063] Entire device

[0064] FIG. 1 illustrates an apparatus (30) for forming a droplet interface and performing an experiment thereon. The apparatus (30) includes a reader (32) and a cartridge (33) that can be inserted into the reader (32). The cartridge (33) includes an AM-EWOD element (34), which is an example of an electro-wetting element. The AM-EWOD element (34) is illustrated in FIG. 2 and is further described below.

[0065] The reader (32) and cartridge (33) may be electrically connected together during use, for example by a cable of a connecting wire (42), but various other methods of providing electrical communication (e.g., wireless connection) may be used.

[0066] The reader (32) also includes a droplet preparation system (35) configured to form a droplet (1) containing liquid in a fluid medium (60) in an AM-EWOD device (34) when a cartridge (33) is inserted. Suitable material properties for the droplet (1) and the fluid medium (60) are discussed below. The droplet preparation system (35) may also perform sample preparation to prepare an analyte to be measured, and alternatively, sample preparation may be performed in the AM-EWOD device (34). The sample may be compartmentalized for library preparation or for sequencing.

[0067] The droplet generation system (35) may include a fluid inlet that performs the function of generating droplets within the AM-EWOD element (34) by introducing liquid into the AM-EWOD element (34) from one or more reservoirs. The droplet generation system (35) may be formed, for example, by a conventional fluid element that controls the flow of liquid by electro-wetting. The droplet generation system (35) preferably has the ability to accurately control the volume of the generated droplets (1), typically up to 2% to 3%. Typically, the droplets may each have a volume of 1 nL to 10 μL.

[0068] The device (30) further includes a control system (37) provided to the reader (32). In this example, the control system (37) includes a control electronic device (38) and a storage device (40) capable of storing any data or any application software associated with the system. The control electronic device (38) may include a suitable circuitry and / or processing element, such as a CPU, a microcontroller, or a microprocessor, configured to perform various control operations regarding the control of the AM-EWOD device (34).

[0069] Among other functions for implementing the features of the present invention, the control electronic device (38) may include part of an overall control system (37) capable of executing program code implemented as a control application within a storage device (40). The storage device (40) may be configured as a non-transient computer-readable medium, such as random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), or any other suitable medium. Additionally, while code may be executed by the control electronic device (38) according to an exemplary embodiment, the functions of such a control system may also be performed through dedicated hardware, firmware, software, or a combination thereof.

[0070] As described in more detail below, the control system (37) is configured to perform control of various elements of the device (1), including the control of the droplet manufacturing system (35) to control the application of a driving signal for forming a droplet (1) and manipulating the droplet. In particular, the control system (37) is configured to form one or more systems of two or more droplets (1). Within the system of droplets (1) or each system, one or more droplet interfaces are formed between each pair of droplets (1). The control system (37) may also provide a graphical user interface (GUI) to the user, which enables the user to input program commands, such as droplet operation (e.g., moving a droplet) or calibration operation (e.g., performing calibration), and to display the results of such operation to the user.

[0071] electro-wetting element

[0072] FIGS. 2 and FIGS. 3 illustrate an AM-EWOD device (34).

[0073] As illustrated in FIG. 2, the AM-EWOD device (34) has a first substrate (44) (which is the outermost substrate in FIG. 2 and FIG. 3) together with a thin-film electronic device (46) disposed on the first substrate (44). An array (50) of driving electrodes (48) is supported by the first substrate (46) on the top of the thin-film electronic device (46). The thin-film electronic device (46) is configured to drive the driving electrodes (48).

[0074] The array (50) of the driving electrodes (48) may be an X × Y rectangular array, where X and Y are any integers. The driving electrodes (48) may be formed from, for example, indium tin oxide (ITO) or other transparent metal oxide, or metal or any other electrically conductive material.

[0075] The AM-EWOD device (34) has a first substrate and also includes a second substrate (54) (which is the uppermost substrate in FIG. 2 and FIG. 3) spaced apart from the first substrate (44) by a spacer (56). As further described below, a droplet (1) is placed between the first substrate (44) and the second substrate (54). A single droplet is shown in FIG. 2 and FIG. 3, but generally, multiple droplets (1) are present.

[0076] The layered structure of the AM-EWOD device (34) is best illustrated in FIG. 3, which exemplifies a part of the structure including two driving electrodes (48) supported by a first substrate (44). The driving electrodes (48) may be formed from a patterned layer of a conductive material.

[0077] An insulating layer (61) comprising a layer (62) of an electrical insulating material coated with a hydrophobic material (64) is disposed on a first substrate (44) to cover driving electrodes (48). The hydrophobic material (64) forms the outermost hydrophobic surface of the insulating layer (61).

[0078] The second substrate (54) faces the hydrophobic surface of the insulating layer (61). The second substrate (54) supports a layer (58) of conductive material deposited on the surface of the second substrate (54) facing the insulating layer (61). The layer (58) of conductive material is patterned as described in more detail to form more electrodes.

[0079] The second substrate (54) is coated with a hydrophobic material (68) covering a layer (58) of conductive material and forms an additional hydrophobic surface opposite to the hydrophobic surface of the insulating layer (61).

[0080] The hydrophobic material (64 and 68) can be formed by any suitable material (which may be the same or different), for example, a fluoropolymer.

[0081] A droplet (1) is received in an AM-EWOD element (34) placed within a fluid medium (60). The droplet (1) and the fluid medium (60) are placed on a hydrophobic surface of an insulating layer (61) and on another hydrophobic surface of a hydrophobic material (68) coating a second substrate (54). In this way, the droplet (1) is interposed between the first substrate (44) and the second substrate (54), which constrains the shape of the droplet (1). This improves the degree of control of the droplet (1) by a driving signal applied to the driving electrodes (48) in the manner described below.

[0082] The droplet (1) has a contact angle (66) with the hydrophobic surface of the insulating layer (61). The contact angle (66) is the surface tension component (Γ) from the hydrophobic surface of (1) to the liquid of the droplet (1). SL ) interface and (2) surface tension component (Γ) from the liquid of the droplet (1) to the surrounding fluid medium (60) LG The interface of ) and (3) the surface tension component (Γ) from the hydrophobic surface to the surrounding fluid medium (60). SG ) is determined by maintaining the balance of the interface. When no voltage is applied, the contact angle (66) satisfies Young's law, and the equation cos θ = ((Γ SG - Γ SL ) / Γ LG It is the size θ given by ).

[0083] Accordingly, when a driving signal is applied to the driving electrodes (48), the driving electrodes (48) can electro-wet the droplet (1). The driving signal generates power that imparts energy to the droplet (1) by effectively controlling the hydrophobicity of the hydrophobic surface of the insulating layer (61). In this energy-gaining state, the droplet (1) will have a deformed shape compared to a lower energy state, that is, a state where the driving signal provides little or no energy to the droplet (1).

[0084] These criteria for the deformed shape may refer to a shape in the plane of the AM-EWOD element (34), that is, a shape parallel to the hydrophobic surface of the insulating layer (61). Although the energy supplied by the driving signal will deform the three-dimensional shape of the droplet (1), the shape is most significantly affected in the plane of the AM-EWOD element (34), and this plane is the direction in which the driving electrodes (48) are arrayed.

[0085] Similarly, a criterion for the shape being deformed compared to the case in a lower energy state may refer to the shape of the contact line of the droplet (1). In this context, the term “contact line” has the general meaning of a line along which the interface between the droplet (1) and the fluid medium (60) contacts the hydrophobic surface of the insulating layer (61) on the driving electrodes (48).

[0086] By selective control of the pattern of the driving signal applied to the selected driving electrodes (48), the droplet (1) can be manipulated and moved in the lateral plane between the first substrate (44) and the second substrate (54). In general terms, such manipulation of the droplet (1) in this manner may apply techniques known for EWOD devices.

[0087] The driving signal may take any form suitable for electro-wetting the droplet (1). Typically, the driving signal may be an AC driving signal, but generally, these signals may also be a DC voltage potential relative to a reference voltage. While the driving signal is being applied, a reference signal is applied to a reference electrode (59) somewhere within the AM-EWOD element, as described further below.

[0088] The reference signal may take any suitable form. In one example, the reference signal may be a fixed reference voltage. In another example where the driving signal is an AC driving signal, the reference signal may be an AC reference signal that is anti-phase with respect to the AC driving signal. In this example, the magnitude of the potential difference between the driving electrodes (48) and the reference electrode (59) is increased, for example, to twice the magnitude when the AC driving signal and the AC reference signal are of the same magnitude compared to the reference signal which is a fixed reference voltage.

[0089] FIG. 4a illustrates a simplified circuit diagram of an electrical load (70A) between a driving electrode (48) and a reference electrode (59) when a droplet (1) is present. The droplet (1) can typically be modeled as a resistor and a capacitor existing in parallel. Typically, the resistance of the droplet (1) is relatively low (e.g., if the droplet contains ions), and the capacitance of the droplet is relatively high (e.g., if the droplet (1) is aqueous, the relative permittivity of the polar liquid is relatively high, for example, about 80). In many situations, the droplet resistance is relatively small, so at the frequency of interest for electro-wetting, the droplet (1) can effectively act as an electrical short circuit. The hydrophobic materials (64 and 68) have electrical characteristics that can be modeled as capacitors, and the insulating material of the layer (62) can also be modeled as a capacitor. The total impedance between the driving electrode (48) and the reference electrode (59) can be approximated by a capacitor, the value of which is typically dominated by the contribution of the insulating material of the layer (62) and the contribution of the hydrophobic material (64 and 68), and the typical layer thickness and material for this may be about pico-Farad value.

[0090] FIG. 4b illustrates a circuit diagram of an electrical load (70B) between a driving electrode (48) and a reference electrode (59) in the case where no droplet (1) is present. In this case, the droplet component is replaced by a capacitor representing the capacitance of a non-polar fluid (60) occupying the space between the top and the first substrate. In this case, the total impedance between the driving electrode (48) and the reference electrode (59) can be approximated by the capacitor, the value of which is typically dominated by the capacitance of the non-polar fluid and is typically small, approximately femto-Farad.

[0091] To drive and sense the driving electrodes (48), the electrical loads (70A and 70B) effectively act as capacitors in their entirety, and their value depends on whether a droplet (1) is present on the given driving electrode (48). If a droplet is present, the capacitance is relatively high (typically about pico-parrad), whereas if a droplet (1) is not present, the capacitance is low (typically about femto-parrad). If the droplet partially covers the given electrode (48), the capacitance can roughly indicate the extent of coverage of the driving electrode (48) by the droplet (1).

[0092] FIG. 5 illustrates an arrangement of thin-film electronic devices (46) in an AM-EWOD device (34). The thin-film electronic devices (46) are located on a first substrate (44) and include an active matrix arrangement of array elements (51), each array element (51) including an array element circuit (72) for controlling the electrode potential of a corresponding driving electrode (48). An integrated row driver (74) and column driver (76) circuit is also implemented in the thin-film electronic devices (46) to supply a control signal to the array element circuit (72). In this way, the array element circuit (72) can selectively perform a function under the control of a control system (37) and actuate the driving electrode (48) to apply a driving signal to the driving electrode (48). Accordingly, the control system (37) controls the driving signal applied to the driving electrodes (48), such as the voltage and timing signal required to perform droplet manipulation operation.

[0093] FIG. 6 illustrates an array element circuit (72) present in each array element (51). The array element circuit (72) includes an input enable, data and actuate, and a driving circuit (88) having an output connected to a driving electrode (48).

[0094] A series of interfaces (82) may also be provided to process a series of input data streams and to facilitate the programming of the required voltage from the array (50) to the driving electrodes (48). The voltage supply interface (84) provides the corresponding supply voltage, the second substrate drive voltage, and other required voltage inputs. The number of connecting wires (86) between the first substrate (44) and the external control electronics, power supply, and any other components can be made relatively small even for large array sizes. Optionally, a series of data inputs can be partially parallelized. For example, if two data input lines are used, the first line can supply data for columns 1 to X / 2 and the second line can supply data for columns (1+X / 2) to M, with minimal modification to the column driver circuit (76). In this way, the speed at which data can be programmed into the array element (51) is increased, which is a standard technique used in liquid crystal display driving networks.

[0095] Droplet interface detection

[0096] FIG. 7 illustrates a method in which a layer (58) of a conductive material is patterned to form a sensor electrode (100), a conductive track (101), and an additional electrode (102), and these electrodes are thus deposited on a second substrate (54) and supported by it.

[0097] The sensor electrode (100) is provided to form an electrical connection with each droplet (1). Providing the sensor electrode (100) is a convenient and reliable method of performing electrical measurements between droplets (1), for example, across a droplet interface formed between droplets, to form an electrical connection with the droplets (1). In contrast, this type of electrical connection is not possible from the driving electrodes (58) due to the presence of an insulating layer (61) containing a layer (62) of electrical insulating material between the driving electrodes (48) and the droplet (1).

[0098] A hydrophobic material (68) covering a layer (58) of a conductive material coating a second substrate (54) is provided with an opening (69) that exposes a portion of the sensor electrode (100), but more generally, the opening is larger and can expose the entire sensor electrode (100). The opening (69) in the hydrophobic material (68) helps to make an electrical contact between the sensor electrode (100) and the droplet (1). The liquid of the fluid medium (60) and / or the droplet (1) flows into the opening (69) and has a lower electrical impedance than the hydrophobic material (68), so as to provide a conductive path.

[0099] These openings (69) may have the additional advantage of acting as hydrophilic paths to help hold the droplet (1) in place when the electrode is de-driven or the element is de-powered.

[0100] These openings (69) can be created by selective removal of hydrophobic material (68), for example by a dry etch process or a lift-off process.

[0101] However, the opening (69) is not essential, and instead, the electrical connection between the sensor electrode (100) and the droplet (1) can be made through a hydrophobic material (68), which may have a sufficiently low impedance (real or virtual) through which electrical measurements can still be taken. In this case, the thickness and material properties of the hydrophobic material (68) are selected accordingly.

[0102] Sensor electrodes (100) are arranged in sets, and each set of sensor electrodes (100) is formed with a size and shape to form an electrical connection with droplets (1), and droplet interfaces are formed between droplets in each system of droplets (1). This can be achieved by an area of ​​sensor electrodes (100) similar to the area enclosed by the contact lines between droplets (1) and sensor electrodes (100), and a distance similar to the distance between the centers of droplets (1) within the formed droplet system, which is the distance between the centers of the sensor electrodes (100) within each set. Each set of sensor electrodes (100) is aligned with each system of droplets (1) so as to form an electrical connection with each droplet (1) within the corresponding system of droplets (1). Accordingly, the control system (37) may be configured to form a plurality of droplet systems in which two or more droplets (1) form a system, wherein each system of droplets (1) is aligned with each set of sensor electrodes (100).

[0103] For example, FIG. 7 illustrates a set of three sensor electrodes, each set comprising two sensor electrodes (100), and a system of three droplets, each system comprising two droplets (1) formed by alignment with the sensor electrodes (100) of each set. However, generally, there may be any number of sets of sensor electrodes (100), and the sets may include any number of sensor electrodes (100) depending on the number of droplets (100) included in each system.

[0104] As a result of this configuration, systems of droplets (1) can be formed in parallel, and experiments can be performed in parallel using each of the sets of sensor electrodes (100) on them. Generally, any number of droplets (2), for example, more than 2, up to a large number of about tens of thousands of systems can be formed.

[0105] A conductive track (101) is connected to a sensor electrode (100) and extends to the edge of a layer (58) of a conductive material, where electrical connection is made to a droplet interface sensor system (110) described further below. Thus, the conductive track (101) provides electrical connection from the sensor electrode (100) to the droplet interface sensor system (110).

[0106] The additional electrode (102) extends around the sensor electrode (100) and the conductive track (101).

[0107] The additional electrode (102) can function as a reference electrode (59) in the circuit diagrams shown in FIGS. 4a and 4b. In this case, the control system (37) is connected to the additional electrode (102) and is configured to apply a reference signal to the additional electrode (102) while applying a driving signal to the driving electrodes (48).

[0108] However, the additional electrode (102) is not essential. If the additional electrode (102) is absent, or even if the additional electrode (102) is present, different electrode(s) may serve as the reference electrode (59). In one example, the sensor electrode (100) may serve as the reference electrode (59).

[0109] In another example, the reference electrode (59) may be provided at any location between the first substrate (44) and the second substrate (54), for example, as a separate element, such as an in-plane reference electrode. In any such example, a control system (37) is connected to the reference electrode (59), for example, sensor electrodes (100), and is configured to apply a reference signal to the reference electrode (59) while applying a driving signal to the driving electrodes (48). In this arrangement, the unactuated driving electrodes (48) on the first substrate (44) can act as a reference, and the droplet (1) can be moved on the second substrate (54) without requiring a reference electrode.

[0110] The reader (32) further comprises a droplet interface sensor system (110) including a measurement unit (111) connected to a sensor electrode (110), and performs electrical measurements between the sensor electrodes (110) electrically connected to each droplet (1) across the droplet interface formed between the droplets. The measurement unit (111) is typically controlled to perform electrical measurements while no driving signal is applied to the driving electrodes (48). This has the advantage of reducing the risk of the measurement signal affecting the electrical measurement by, for example, physically affecting or damaging the droplet system being measured, or by causing electrical interference with the measurement unit (111).

[0111] The elements of the thin-film electronic device (46) are electrically insulated from the sensor electrodes (100) and the measurement unit (111) and do not participate in the performance of electrical measurements.

[0112] Any suitable electrical measurement, for example, impedance, current, or capacitance measurement, may be performed. In a possible configuration, the electrical measurement may be performed by applying a voltage through another sensor while one of the sensor electrodes (100) is grounded, and measuring the current supplied therethrough. Thus, the actual and virtual parts of the electrical impedance of the droplet interface (2) can be determined.

[0113] The measurement unit (111) may be formed by electronic components suitable for droplet interface experiments, such as electronic components including a detection channel including an amplifier array. In one example, the measurement unit (111) may include a patch clamp array. In another example, the measurement unit (111) may have the same configuration as the signal processing function described in International Publication WO-2011 / 067559.

[0114] Electrical measurements can be performed in a frequency range from a lower limit of 1 Hz, 10 Hz, or 100 Hz to an upper limit of 10 MHz, 100 KHz, or 10 KHz, or in any combination thereof.

[0115] The measurement unit (111) may be configured to apply a potential difference between each pair of sensor electrodes (100) on which the measurement is performed while performing these measurements.

[0116] The measurement unit (111) is controlled by the control system (37) to perform electrical measurements from any system of the droplet (1) after the droplet (1) is formed in the AM-EWOD element (34) under the control of the control system (37).

[0117] The electrical measurement may be of any suitable type, for example, an impedance measurement and / or a measurement of ion current flow across the droplet interface. Where the measurement is performed across a droplet interface comprising a membrane of amphiphilic molecules with inserted through-membrane pores, the electrical measurement may be, for example, a measurement of ion current flow between droplets through the through-membrane pores and / or an electrical measurement that depends on the analyte interacting with the through-membrane pores.

[0118] The measurement may be an optical measurement disclosed in, for example [Soni GV et al., Rev Sci Instrum. 2010 Jan;81(1):014301 and T Gilboa and A Meller, Analyst, 2015,140, ​​4733-4747], or a combination of optical and electrical measurements.

[0119] The droplet interface sensor system (110) may further include an analysis system (112) configured to process electrical measurements that vary depending on the analyte interacting with the membrane-penetrating pores in order to analyze the analyte. For example, if the analyte is a polymer containing polymer units, the analysis system may be configured to process electrical measurements to derive the estimated identity of the polymer units of the polymer. The analysis system (112) may process electrical measurements using any suitable known technique, some examples of such techniques are further described below.

[0120] The analysis system (112) may be formed by a suitable combination of (1) a hardware stage for pre-processing electrical measurements supplied as signals from a measurement unit (111), such as a field-programmed gate array (FPGA), and (2) a processor for processing signals supplied from said hardware stage. The processor may be any suitable type of processing element. The processor may be implemented within a reader (32) as shown in FIG. 1 and may execute software that can be stored in a storage device (40). Alternatively, the processor may be implemented by a processing element outside the reader (32), such as a conventional computer device.

[0121] For example, the measurement unit (111) and the analysis system (112) may have the same configuration as the signal processing function described in International Publication WO-2011 / 067559.

[0122] The droplet interface sensor system (110) may be combined with other types of measurement systems to perform measurements, for example, capacitance measurements from driving electrodes, measurements from additional electrodes (not shown), and / or, without limitation, electromagnetic radiation using absorbed or emitted infrared, ultraviolet, and / or fluorescence resonance energy transfer (FRET) using a technique that may utilize a dye or antibody labeled with the technique.

[0123] Droplet detection

[0124] The array element circuit (72) may also include a droplet sensor circuit (90), which is in electrical communication with the driving electrode (48). The droplet sensor circuit (90) provides a sensing capability to detect the presence or absence of a droplet (1) at the location of each driving electrode (48). In this way, the array element circuit (72) may also perform the function of detecting the presence or absence of a droplet (1) at the location of the array element (51) during the operation of the droplet (1). However, due to the presence of an insulating layer (61) including a layer (62) of electrical insulating material between the driving electrodes (48) and the droplet (1), it may be difficult or cumbersome to perform electrical measurements suitable for studying the droplet interface, or the processes occurring at the droplet interface.

[0125] The droplet sensor circuit (90) may, for convenience, utilize capacitive sensing using an impedance sensor circuit. The droplet sensor circuit (90) may include an impedance sensor network of a type known in the art, such as as described in, for example, U.S. Patent US-8,653,832 and UK Patent GB-2,533,952. As described therein, the droplet (1) may be driven by electro-wetting and may be detected by a capacitive or impedance sensing means. Typically, the capacitive and impedance sensing may be similar and may be performed simultaneously or nearly simultaneously on all array elements (51). By processing the information returned from these sensors (e.g., in the application software of the storage element (40) of the reader (32)), the control system (37) may determine the location, size, centroid, and perimeter of each droplet (1) present in the AM-EWOD element in real time or near real time.

[0126] Alternatively, such detection may be performed by some other means, for example, optical means or thermal means. An alternative to the droplet sensor circuit (90) is to provide an external sensor, such as an optical sensor, that can be used to detect droplet characteristics, as is known in the field of electro-wetting devices.

[0127] The control system (37) generates and outputs a control signal for the droplet sensor circuit (90) to perform a detection operation during the operation of the droplet (1). The integrated sensor row addressing (78) and column detection circuit (80) are performed in the thin film electronics (46) for addressing and reading the droplet sensor circuit (90) in each array element circuit (72). Typically, the reading of the droplet sensor circuit (90) may be controlled by one or more addressing lines (e.g., RW) that may have one or more outputs, e.g., OUT, which may be universal to the array element (51) in the same row of the array (50) or universal to all array elements (50) within the same column of the array (50).

[0128] The control system (37) can use the output of the droplet sensor circuit (90) to control the timing of applying a driving signal to the driving electrodes (48) when operating the droplet (1).

[0129] Formation of droplet interface

[0130] The control system (37) is configured to control the AM-EWOD element (34) to form a droplet (1) system having one or more droplet interfaces (2) between pairs of droplets (1) as follows.

[0131] First, the control system (37) controls the droplet manufacturing system (35) to form the droplet (1) in the AM-EWOD element (34) if necessary for each system of the droplet (1). The droplet (1) can be manufactured from any suitable reagent if necessary for the experiment being performed. Suitable reagents are listed below.

[0132] Next, the control system (37) controls the application of a driving signal to the driving electrodes (48) to form a system of droplets (1).

[0134] It was considered to simply apply a driving signal to manipulate the droplets (1) by simply moving the droplets across an array (50) of driving electrodes (48) that are formed and contact each other. However, in this case, the droplets (1) tend to fuse, and it is difficult to maintain a droplet interface between the droplets (1). Optimizing the conditions to promote the formation of the droplet interface (2) is difficult because it depends on several factors that electro-wetting tends to vary between samples, such as salt concentration, droplet reagent (particularly membrane component), and droplet size.

[0135] Therefore, as described below, another method using two steps is implemented.

[0136] An example of this method is illustrated in FIGS. 8 through 12, which sequentially show a plan view of a pair of droplets (1) on an array (50) of driving electrodes (48) as the droplets are manipulated during this method. In particular, FIGS. 8 through 12 illustrate the contact lines of the droplets on the array (50) of driving electrodes (48). FIGS. 8 through 12 also illustrate the pattern (53) of the applied driving signal by hashing the selected driving electrodes (48) to which the driving signal is applied (53). This example is for illustrative purposes only and is not limiting. Various changes, such as the size of the droplets (1) and the pattern of the driving signal, may be made. Additionally, FIGS. 8 through 12 relate to an example in which the liquid of the droplet (1) is polar and the fluid medium (50) is nonpolar, and the result is that the driving electrodes (48) to which the driving signal is applied are electro-wet. In a conceptual alternative where the liquid of the droplet (1) is nonpolar and the fluid medium (50) is polar, the pattern of the driving signal will be reversed, and the result is that the driving electrodes (48) to which the driving signal is not applied attract the nonpolar droplet.

[0137] As background, in the relaxed state of the droplet (1) which is not electro-wetting by the application of a driving signal to the driving electrodes (48), the droplet (1) will take the shape of the lowest surface energy, which is generally a circular shape having a uniform characteristic of the hydrophobic surface of the insulating layer (61).

[0138] In the first step of this method, a driving signal is applied to selected driving electrodes (48) to provide energy to one of the two droplets (1), or preferably both, between which a droplet interface is formed. For clarity of explanation, the case where energy is provided to both of the two droplets (1) is described from now on.

[0139] The shape of the droplets (1) in the energy-gaining state is deformed compared to the shape of the droplets (1) in a lower energy state. In this energy-gaining state, two droplets (1) are moved in close proximity with a gap (3) between them. At this time, the droplets (1) do not come into contact with each other due to the gap (3).

[0140] The first step of the above method can be performed under feedback control from the droplet sensor circuit (90).

[0141] Examples of the process applied in the first step are illustrated in FIGS. 8 to 10 as follows.

[0142] FIG. 8 illustrates a step in which a pattern (53) of a driving signal is applied to a square 4×4 group of driving electrodes (48). This forms a predetermined corresponding shape that minimizes the surface energy of the droplets (1) by imparting energy to two droplets (1), which is generally square but has slightly rounded corners. FIG. 8 also illustrates a method in which the droplets (1) can be moved together. In particular, FIG. 8 illustrates the case where the pattern (53) of the driving signal is applied to a group of driving electrodes (48) that is shifted compared to the previous step. This results in moving the droplets (1) toward the group of driving electrodes (48) in the direction of the arrow (4). In this way, the droplets (1) can be shaped and moved.

[0143] FIG. 9 illustrates a step in which a pattern (53) of a driving signal is applied to a rectangular 2×8 group of driving electrodes (48). This forms a predetermined corresponding shape that minimizes the surface energy of the droplets (1) by imparting energy to the two droplets (1), which is generally rectangular but has slightly rounded corners. At this step, the rectangular 2×8 group of driving electrodes (48), and thus the droplets (1), come into close proximity to the gap (3) between the two columns of the driving electrodes (48).

[0144] FIG. 10 illustrates a step subsequent to the step illustrated in FIG. 9, in which the driving signal has the same pattern (53), except that one of the 2×8 groups of driving electrodes (48) is shifted by one column of driving electrodes (48), so that the rectangular 2×8 groups of driving electrodes (48), and thus droplets (1), come into close proximity to the gap (3) of a single column of driving electrodes (48). In this example, this is the final step of the first step of the method.

[0145] In the second step of this method, the applied driving signal is changed so that the energy of the droplet (1) is lowered to a lower energy state.

[0146] At this stage, the change is preferably to not apply any driving signal to the driving electrodes (48) affecting the droplets (1). In this case, no energy is supplied to the droplets (1) from the driving electrodes (48), so the lower energy state is the minimum energy state of the droplets (1), where the shape of these droplets (1) after relaxation is influenced only by material properties. Alternatively, the change may, in principle, be to apply a driving signal that imparts energy to the droplets (1) to a smaller degree, so that the droplets (1) are relaxed and the shape of these droplets changes to a smaller degree than in the case where no driving signal affecting the droplets (1) is applied to the driving electrodes (48).

[0147] As a result of being placed in a lower energy state, the surfaces of the droplets (1) facing each other across the gap (3) relax into the gap (3) and come into contact with each other, thereby forming a droplet interface (2) between the two droplets (1). Thus, the movement of the droplet (2) surface is induced by the relaxation from the energy-gaining state of the droplets (1) generated in the first step. This is a passive process that provides reliable formation of the droplet interface (2). The rate at which the droplets (1) relax may depend on one or more factors, such as the relative viscosity of the liquid of the droplets (1) with respect to the fluid medium (50), the size of the droplets (1), and / or the size of the gap (3).

[0148] The geometric characteristics of the device (size of the droplet (1), height of the gap between hydrophobic surfaces, etc.) and the surface tension at the droplet interface (2) depend on the choice of material and material properties, and when the surfaces of the droplets (1) come into contact, the droplets (1) are not fused or aggregated, but rather are arranged so that a droplet interface (2) is formed. The geometric characteristics of the pattern of the driving signal and the spatial dimensions of the droplet (1) will typically be arranged so that the droplet interface (2) is formed with a minimum surface area.

[0149] The applied driving signal may be modified in any way to de-energize the droplet (1). If the driving signal applied to electro-wet the driving electrodes is an AC driving signal, the modification is preferably to replace the AC driving signal that energizes the droplet (1) by a DC potential, e.g., ground potential or floating potential. This has the benefit that the AC signal is no longer applied to the driving signal, which contributes to the formation of the droplet interface (2) because the presence of the AC electric field due to the AC signal increases the risk of the droplet interface (2) rupturing and the surfaces of the droplets (2) coming into contact.

[0150] Alternatively, other modifications to remove energy from the droplet (1) may be made. An alternative is to remove all power from the array (50) of driving electrodes (48). However, it may be desirable to apply a DC potential to help shield the droplet interface (2) from unwanted environmental electromagnetic interference.

[0151] The inventors understood that it is desirable not to de-drive the droplet in the conventional method by applying an AC voltage waveform, because the generated perturbation can damage the droplet interface (2) or interfere with electrical measurement through the droplet interface (2).

[0152] An example of the process applied in the second stage is presented in FIGS. 11 and FIGS. 12 as follows.

[0153] FIG. 11 illustrates a step in which the pattern of the driving signal changes compared to the pattern shown in FIG. 10 by stopping the application of the driving signal to two 2×8 groups of driving electrodes (48) and instead applying a DC potential or floating potential. FIG. 11 illustrates the droplet (1) at the moment the change occurs, where the droplet immediately has the same generally rectangular shape as before. However, the droplet then relaxes to a lower energy state shown in FIG. 12. In the absence of other droplets (1), each droplet (1) will take its own lower energy state, and while this energy state is generally circular, the center of mass of the droplet (1) generally remains in the same location. Thus, when relaxing toward having a lower energy state, the surfaces of the droplets (1) facing each other across the gap (3) relax into the gap (3) and come into contact with each other, forming a droplet interface (2).

[0154] In the energy-gaining state illustrated in FIGS. 8 to 10, the specific shape of the droplet (2) is not limited, and any generally arbitrary shape that allows the droplet (1) to relax and come into contact to form a droplet interface may be used. Typically, the shape of the contact line of the energy-gaining droplets (2) may be elongated, where the gap (3) extends along the length of the elongated shape. Any elongated shape, for example, a rectangular shape as shown in FIG. 9, an ellipsoidal shape, or a more complex shape may be selected. A non-elongated shape, for example, a square shape as shown in FIG. 8, may be used.

[0155] The exact shape of the droplet (1) is selected by controlling the pattern of the driving signal, but it may vary due to the surface tension between the droplet (1) and the fluid medium (50), which varies depending on the material properties.

[0156] When the shape of the contact line of the energy-receiving droplets (2) is elongated, the shape of the energy-receiving droplet (1) may have an aspect ratio of at least 2:1, preferably at least 4:1 or at least 8:1. Generally, increasing the aspect ratio helps the droplets (1) make contact by increasing the degree of movement of the surface of the droplets (1) when energy is removed from the droplets.

[0157] In the first step, the gap (3) between the droplets (1) is selected to form a droplet interface (2) when the two droplets (1) are sufficiently close but not in contact, so that these droplets are placed in a lower energy state. When the droplets (1) come close, the width of the gap (3) is selected to bring the droplets (1) into contact when the driving signal changes. This may vary depending on the droplets (1) in the energy gain state. Typically, the width of the gap (3) when the two droplets (1) come close may be selected so that the centroids of the two droplets (1) are separated by a distance shorter than the combined radius of the droplets (1) along the line between the two centroids of the droplets (1) in the lower energy state.

[0158] The gap (3) may have the width of a single row or column of driving electrodes (38) in the array (50), or two or more rows or columns of driving electrodes (38) in the array (50).

[0159] To facilitate these processes, the area enclosed by the contact line of the droplet (1) is preferably larger than the area of ​​the driving electrodes (38). This increases the resolution of shape control in the droplet (1) in the energy-gaining state, thereby helping to enable movement of the droplet (1) across the array and movement to contact of the surface of the droplet (1) during relaxation. Accordingly, the AM-EWOD device (1) is designed to have driving electrodes (48) sized with respect to the typical size of the droplet (1) desired for experimental use. A specific advantage of the active matrix array is that it enables the application of driving patterns of driving signals at high resolution relative to the size of the droplet (1).

[0160] Typically, the area surrounded by the contact line of the lower energy state droplet (1) is at least twice, preferably at least five times, at least ten times, or at least twenty times the area of ​​the driving electrode (48). Thus, the area surrounded by the contact line of the lower energy state droplet (1) can cover at least two driving electrodes (48), preferably at least five driving electrodes (48), at least ten driving electrodes (48), or at least twenty driving electrodes (48).

[0161] The above description refers to applying energy to both droplets (1) for ease of explanation, but alternatively, the droplet interface (2) may be formed by applying energy to only one of the two droplets (1) in the first step. In this case, the surface of the one droplet (1) relaxes while changing the driving signal in the second step and comes into contact with the stationary surface of the other droplet (1).

[0162] Droplet system

[0163] In the above description, a single droplet interface (2) is formed between two droplets (1) of a system. Using a similar method, multiple droplet interfaces (2) may be formed between each pair of droplets within a system consisting of three or more droplets (1). Droplet interfaces (2) may be formed sequentially by contacting the droplets (1) continuously or simultaneously. For example, FIG. 13 illustrates an example of forming a system of three droplets (1) having two droplet interfaces (2) formed sequentially, and FIG. 14 illustrates an example of forming a system of three droplets (1) having two droplet interfaces (2) formed simultaneously. In each of these examples, the droplet interfaces (2) are formed using the method described above.

[0164] Generally, the configuration of the formed droplet system of droplets (1) is selected in a manner that allows for the desired experiment to be performed. In such a formed system, the droplets (1) may be arranged in series, for example, as shown in the system of three droplets (1) illustrated in FIGS. 13 and 14, and the droplet interfaces (2) may also be arranged in series. Alternatively, in the formed system, the droplets (1) may have a more complex arrangement or cluster, two non-limiting examples of which are illustrated in FIGS. 15 and 16.

[0165] In this system of droplets (1), the droplets (1) may have the same or different volumes, and the droplets (1) may have the same or different components.

[0166] One or more droplets in the system may include a membrane-penetrating pore that can be inserted into the droplet interface (2). Typically, after the droplet interface (2) is formed, the membrane-penetrating pore is spontaneously inserted into the droplet interface (2), and an electrical measurement can subsequently be performed. One or more droplets (1) in the system may include an analyte that interacts with the membrane-penetrating pore.

[0167] In particular, when manufacturing a system of three or more droplets (1) having multiple droplet interfaces (2), there are many advantages in the use of the device (1) for forming droplet interfaces (2) and subsequently performing electrical measurements on the droplet interfaces (2) thus formed. For example, a relatively small sample volume can be used compared to some other techniques involving the formation of an array of planar films. This enables the use of long length polynucleotides, as the chance of shearing is reduced as library manufacturing can occur on the same device as the measurement. Considering that the sample does not need to be moved, the risk of contamination is lower. Since all samples are contained in one or both of the droplets (1), there is less sample loss that can be recovered. Since electro-wetting is used for all liquid operations, the need for pumps or other moving parts is eliminated. Since electro-wetting positioning is controlled via a programmed script, sample manufacturing can be automated. Afterwards, the desired components, including polymer vesicles, reagents, voids, and the analyte, can be added to the droplet of the DNA sample.

[0168] In one type of experiment, the droplet (1) is periodically divided from the volume of the sample to monitor, for example, ongoing reactions occurring in the sample. This provides time, titration of reactants, changes in conditions, etc. for analysis.

[0169] Other advantages include the following:

[0170] · Ability to perform library preparation / PCR coupled with detection / sequencing; ease of using libraries for sequencing (automated, walk-away)

[0171] · Low pollution risk

[0172] · Use of compartmentalized samples for library or sequencing

[0173] · Enables sampling of different locations of the sample / reaction, e.g., length through a gel / mesh / diffusion barrier, location on the cell sample, and / or concentration / thermal / density gradients.

[0174] Feedback and transformation

[0175] As described above, the device (1) is suitable for forming a droplet interface (2) in the system of droplets (1) and for conducting experiments on the droplet interface (2). A specific advantage is obtained by a control system (37) that modifies the formed droplet system of droplets (1) in response to the output of the droplet interface sensor system (110). Thus, the system of droplets (1) can be modified to modify the performance of the ongoing experiment using feedback from previously performed experiments. This provides a powerful experimental tool because the experiment can be performed adaptively.

[0176] Various outputs of the droplet interface sensor system (110) can be used to provide feedback, for example as follows.

[0177] The output of the droplet interface sensor system (110) that can be used includes an electrical measurement performed by a measurement unit (111). This provides a first type of control. Since electrical characteristics are fundamental to related processes such as the formation of a droplet interface and reactions occurring at the droplet interface, this first type of control can allow these processes to be taken into account and adaptively modified. For example, the electrical measurement performed by the sensor system can be used to determine whether the droplet interface (2) has been successfully formed.

[0178] The output of the droplet interface sensor system (110) that can be used includes the output of the analysis system (112). This provides a second type of control. Since this analysis allows for obtaining a higher level of information regarding, for example, the sample being analyzed, this second type of control provides powerful experimental adaptation based on the analysis results.

[0179] The control system (37) can modify the formed droplet system of the droplets (1) in various ways, for example as follows.

[0180] The control system (37) can modify the formed droplet system of droplets (1) by separating the droplet interface (2) within the droplet system. To do this, the control system (37) applies driving signals of a predetermined pattern to the driving electrodes (48) to separate one or both of the droplets formed between the droplet interface (2). The separation of the droplets (1) separates the droplet interface (2).

[0181] This separation can be used, for example, to stop interactions occurring at the droplet interface (2). This can be done, for example, when an electrical measurement performed by the measurement unit (111) indicates that the droplet interface (2) was not successfully formed, or when the output of the analysis system (112) indicates that the analysis is complete, for example, because the analyte has been reduced or because sufficient electrical measurements have been performed on a specific analyte.

[0182] The control system (37) can modify the formed droplet system of droplets (1) by bringing a new droplet (1) into contact with the current droplet (1) in the droplet (1) system to form a droplet interface (2) between the new droplet (1) and the current droplet (1). To do this, the control system (37) applies a driving signal to the driving electrodes (48) using the same method as described above.

[0183] This formation of a new droplet interface (2) can be used, for example, when an electrical measurement performed by a measurement unit (111) indicates that the droplet interface (2) has not been successfully formed and that a new droplet interface is desired, or when the output of an analysis system (112) indicates that the analysis at the droplet interface (2) has been completed and that additional measurements are desired.

[0184] The control system (37) can modify the formed droplet system of droplets (1) by bringing a new droplet (1) into contact with the current droplet (1) in the droplet (1) system and fusing the new droplet (1) and the current droplet (1). To this end, the control system (37) applies a driving signal to driving electrodes (48) that move the new droplet (1) to contact the current droplet (1) and fusing them. Fusion of the droplets (1) can be achieved simply by moving the new droplet (1) to contact the current droplet (1) without using the method described above to form a droplet interface. Alternatively or additionally, fusion of the droplets (1) can be achieved by applying an AC driving signal that ruptures the droplet interface to be formed between the new droplet (1) and the current droplets.

[0185] This fusion of the new droplet (1) of the system and the current droplet (1) can be used, for example, to introduce a new reagent into the current droplet (1) when one member of the redox couple in one of the droplet (1) pairs is reduced.

[0186] When a new droplet (1) is fused in this way, it may be possible for the new droplet (1) not to contain amphiphilic molecules at the interface between the liquid of the droplet (1) and the fluid medium (50).

[0187] These and other methods of modifying the formed droplet system of droplets (1) can be used together in any combination, for example, to perform a multi-stage experiment.

[0188] Although some specific applications are listed above, they are non-limiting, and in fact, one of the benefits of feedback is this versatility. Non-limiting examples of some additional applications are as follows:

[0189] · Automated insertion of membrane-penetrating voids

[0190] · Adaptation of the droplet (1) system to the unwanted insertion of voids or secondary voids

[0191] · Control based on reaction / sample conditions

[0192] · Promotion of separation at the droplet interface

[0193] · Delivery of more samples or reagents

[0194] · Delivery of different samples

[0195] · Delivery of more mediators to the droplets

[0196] · Separation of droplets (1) for taking the sample anywhere and / or recovering the sample and / or returning the sample to its original volume

[0197] · Changes in reaction conditions (e.g., temperature, additives, quenching / activation)

[0198] · Performance of alternative measurements (e.g., absorbance)

[0199] · Return of the sample to its original volume

[0200] · Performance of a new reaction on the analyzed sample (or part thereof)

[0201] · Control of multiple pore types and balancing of each of the multiple membranes

[0202] · Formation of a membrane array having multiple membranes and pores interfacing with the same sample

[0203] · Conducting experiments only until sufficient information is obtained, thereby increasing the overall experimental throughput.

[0204] · Sample queuing / pooling, e.g., allowing the transfer of library samples from the sample queue upon request and / or changing the queue order

[0205] · Pooling of samples as a result of sequencing / detection

[0206] · Determination of which multiple samples will be analyzed

[0207] · Determination of duration / success criteria

[0208] · Determination of conditions for sample modification prior to membrane / pore analysis (e.g., type / concentration of library preparations)

[0209] · When the droplet (1) is periodically divided from the sample volume, use of the results of the previous experiment as feedback to adapt the reaction / sample conditions

[0210] · Performance of directed evolution using membranes / pores as sensors

[0211] Droplets in a fluid medium

[0212] Where the present invention refers to a droplet comprising a liquid in a fluid medium, the liquid and the fluid medium may be selected as follows. Generally, any liquid that forms a droplet in a fluid medium may be used, but some possible materials are as follows.

[0213] The fluid medium may, in principle, be a gaseous medium, but is preferably a liquid medium.

[0214] In some cases, and often when the fluid medium is a liquid medium, one of the liquid and the fluid medium is polar, and the other of the liquid and the fluid medium is nonpolar. Preferably, the liquid of the droplet is polar, and the fluid medium is nonpolar.

[0215] If one of the liquid and fluid media is polar, the polar medium is typically an aqueous liquid containing water. The aqueous liquid may additionally contain one or more solutes. The aqueous liquid may additionally contain, for example, a buffer to appropriately control the pH of the aqueous medium, and the aqueous liquid may contain a supporting electrolyte. The aqueous medium may include, for example, a redox couple, or a member of a redox couple that may be partially oxidized or reduced to provide said redox couple. Redox couples are those known in the art, e.g., Fe 2+ / Fe 3+ , ferrocene / ferrocenium or Ru 2+ / Ru 3+ It can be selected from. Examples of these are ferro / ferricianide, ruthenium hexamide, and ferrocene carboxylic acid.

[0216] Alternatively, if one of the liquid and fluid media is polar, the polar medium may include a polar organic solvent. The polar organic solvent may be, for example, a protic organic solvent, such as an alcohol, or the polar organic solvent may be an aprotic polar organic solvent.

[0217] The liquid of the droplet may be any liquid suitable for performing the type of experiment described below. Different droplets may contain different liquids.

[0218] If the other of the liquid and fluid media is nonpolar, the nonpolar medium may contain oil. The oil may be a single compound, or the oil may contain a mixture of two or more compounds.

[0219] For example, oil is a pure alkane hydrocarbon.

[0220] The oil may include, for example, silicone oil. Suitable silicone oils include, for example, poly(phenylmethylsiloxane) and poly(dimethylsiloxane) (PDMS). The silicone oil may include hydroxy-terminated silicone oil, for example, hydroxy-terminated PDMS.

[0221] Additionally or alternatively, the oil may contain a hydrocarbon, e.g., hexadecane, but any suitable hydrocarbon may be used. If the oil contains a hydrocarbon, the oil may contain a single hydrocarbon compound or a mixture of two or more hydrocarbons. If the oil contains a hydrocarbon, the hydrocarbon may be branched or unbranched. The hydrocarbon may be, e.g., squalene, hexadecane, or decane. In one embodiment, the hydrocarbon is hexadecane. However, in some embodiments, the hydrocarbon may be substituted with a halogen atom, e.g., bromine.

[0222] The oil may comprise a mixture of one or more silicone oils and one or more hydrocarbons. Both the silicone oil and the hydrocarbon in the mixture may be as further defined above. The silicone oil may be, for example, poly(phenylmethylsiloxane) or PDMS.

[0223] Other types of oil are also possible. For example, the oil may be a fluorocarbon or bromine-substituted C 10 -C 30 It could be Alcan.

[0224] Amphiphilic molecule

[0225] When one of the liquid and fluid medium is polar and the other is nonpolar, the droplet may additionally contain amphiphilic molecules at the interface between the liquid and fluid medium of the droplet. These amphiphilic molecules serve to stabilize the droplet in the fluid medium prior to the formulation of the droplet interface. Additionally, the amphiphilic molecules may cause the droplet interface to contain a film of amphiphilic molecules when formed.

[0226] Many different types of amphiphilic molecules can be used. Some non-limiting examples of the types of amphiphilic molecules that can be used are as follows.

[0227] In one example, the amphiphilic molecule may include lipids, and these lipids may have a single component or a mixture of components as is customary when forming a lipid bilayer.

[0228] Any lipid forming a membrane, such as a lipid bilayer, may be used. The lipid is selected to form a lipid bilayer having the necessary properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid may comprise one or more different lipids. For example, the lipid may contain 100 or fewer lipids. The lipid preferably contains 1 to 10 lipids. The lipid may comprise naturally occurring lipids and / or artificial lipids.

[0229] Lipids can also be chemically modified.

[0230] Amphiphilic polymer membranes are preferred over lipid membranes due to their ability to withstand higher voltages.

[0231] In another example, the amphiphilic molecule may include an amphiphilic compound comprising a first external hydrophilic group, a hydrophobic core group, and a second external hydrophilic group, wherein the first and second external hydrophilic groups are each connected to the hydrophobic core group.

[0232] Some of these amphiphilic compounds are disclosed in International Publication WO 2014 / 064444.

[0233] Other such amphiphilic compounds are disclosed in US-6,916,488, which discloses many polymeric materials incorporated herein by reference and which can be used as planar amphiphilic membranes in apparatus (1). In particular, triblock copolymers, for example, silicon triblock copolymer membranes, such as poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA), are disclosed.

[0234] Examples of silicon triblock polymers that can be used are 7-22-7 PMOXA-PDMS-PMOXA, 6-45-6 PMOXA-PE-PMOXA, and 6-30-6 PMOXA-PDMS-PMOXA, where the nomenclature refers to the number of subunits.

[0235] These triblock copolymers can be provided in the form of vesicles in droplets.

[0236] Depending on the properties of the amphiphilic molecules, the membrane can be a double layer of amphiphilic molecules or a single layer of amphiphilic molecules.

[0237] Alternatively, amphiphilic molecules can be replaced by other surfactants.

[0238] Different droplet interfaces may include membranes of different amphiphilic molecules, for example, membranes comprising a lipid bilayer and polymer membranes, such as the silicon triblock polymer membrane disclosed in International Publication WO2017 / 004504 as described above.

[0239] The electrical measurements performed can be used to study the membrane of the amphiphilic molecule itself or its interactions, for example, to study the drug-membrane permittivity.

[0240] Membrane penetration void

[0241] Generally, any membrane-penetrating pore that can be inserted into a droplet interface may be used. Different droplets may include the same or different membrane-penetrating pores, so that when multiple droplet interfaces are formed between multiple pairs of different droplets, the same or different membrane-penetrating pores may be inserted into these droplet interfaces.

[0242] Some non-limiting examples of types of membrane-penetrating pores that can be used are as follows.

[0243] A transmembrane pore is a channel structure that provides a path from one membrane to another through which ions can flow. The channel can vary in width along its length and typically has an inner diameter of 0.5 nm to 10 nm.

[0244] Any suitable transmembrane pore may be used in the present invention. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami pores are disclosed in International Publication WO2013 / 083983.

[0245] The transmembrane pore is preferably a transmembrane protein pore.

[0246] Transmembrane protein pores can be monomers or oligomers. The pores can be hexameric, heptmeric, octmeric, or nonmeric pores. The pores can be homo-oligomers or hetero-oligomers.

[0247] Transmembrane protein pores can be derived from CsgG, for example, from CsgG from the E. coli strain K-12 substrain MC4100. Examples of suitable CsgG pores are described in WO-2016 / 034591, WO-2017 / 149316, WO-2017 / 149317 and WO-2017 / 149318.

[0248] Transmembrane protein pores typically contain barrels or channels through which ions can pass and flow. Subunits of the pore typically surround a central axis and contribute strands to transmembrane β-barrels or channels or transmembrane α-helical bundles or channels.

[0249] The barrel or channel of the transmembrane protein pore contains an amino acid that facilitates interaction with the analyte, such as a nucleotide, polynucleotide, or nucleic acid. The pore may be modified, for example, by the substitution or deletion of one or more amino acids.

[0250] The transmembrane protein pores for use according to the present invention may be derived from β-barrel pores or α-helical bundle pores. β-barrel pores comprise barrels or channels formed from β-strands. Suitable β-barrel pores are β-toxins, e.g., α-hemolysin, anthrax toxin, and leukocidin, and other membrane proteins / porins of bacteria, e.g., Mycobacterium smegmatis ( Mycobacterium smegmatis ) porins (Msp), e.g., MspA, MspB, MspC or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria ( Neisseria ) Automatic transport vehicle (Autotransporter lipoproteins (NalP) and other pores, such as lysenin, are included but not limited to these. The α-helical bundle pores include barrels or channels formed from the α-helium. Suitable α-helical bundle pores include inner membrane proteins and α-outer membrane proteins, such as WZA and ClyA toxins, but are not limited to these.

[0251] The transmembrane pores may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, and the hemolytic protein fragaceatoxin C (FraC). The transmembrane protein pores are preferably derived from CsgG, and more preferably from CsgG from the E. coli strain K-12 substrain MC4100.

[0252] Membrane-penetrating pores can be derived from risenin. Suitable pores derived from risenin are disclosed in International Publication WO 2013 / 153359.

[0253] The pores may be variants of the nanopores listed above. The variants may have at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, 97%, or 99% homology based on amino acid similarity or identity with respect to the amino acid sequence.

[0254] Standard methods in the art may be used to determine homology. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology, for example, in its default settings (Reference [Devereux et al (1984) Nucleic Acids Research 12, p387-395]). PILEUP and BLAST algorithms are, for example, referenced in the literature [Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al As described in

[1990] J Mol Biol 215:403-10], it can be used to calculate homology or align sequences (e.g., to identify equivalent residues or corresponding sequences (typically in their default settings)). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). Similarity can be measured by using pairwise identity or by applying a scoring matrix, such as BLOSUM62, and converting to equivalent identity. Since these represent functionality rather than evolutionary changes, sophisticatedly mutated positions can be indicated when determining homology. Similarity can be determined more selectively by applying a position-specific scoring matrix, for example using PSIBLAST, on a comprehensive database of protein sequences. Different scoring matrices reflecting amino acid chemo-physical properties (e.g., charge) rather than the frequency of substitutions over evolutionary time scales may be used.

[0255] Amino acid substitutions may be performed on the amino acid sequence of SEQ ID NO: 3, for example, 1, 2, 3, 4, 5, 10, 20, or 30 or fewer substitutions. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge similar to the amino acids they replace. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid at the site of an existing aromatic or aliphatic amino acid.

[0256] Any protein described herein, such as a transmembrane protein void, may be produced synthetically or by recombinant means. For example, the void may be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the void may include non-naturally occurring amino acids or be modified to increase the stability of the protein. If the protein is produced by synthetic means, such amino acids may be introduced during production. The void may also be altered after synthetic or recombinant production.

[0257] Any protein described herein, such as a transmembrane protein void, may be generated using standard methods known in the art. Polynucleotide sequences encoding voids or constructs may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding voids or constructs may be expressed in bacterial host cells using standard techniques in the art. Voids may be generated in cells by in situ expression of polypeptides from recombinant expression vectors. Expression vectors optionally carry inducible promoters to control polypeptide expression. These methods are described in the literature [Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY].

[0258] Pores can be generated on a large scale from protein-producing organisms after purification by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, Bio-Cad systems, Bio-Rad BioLogic systems, and Gilson HPLC systems.

[0259] analytes

[0260] The droplet may contain an analyte capable of interacting with the membrane-penetrating pore, which is also referred to as the target analyte, template analyte, or analyte of interest. For example, the analyte may be a polymer or a drug.

[0261] The electrical measurements performed may vary depending on the interaction between the analyte and the transmembrane pores. The electrical measurements may be the measurement of ion currents through the pores.

[0262] If the electrical measurement depends on the interaction between the analyte and the penetrating pore, the analysis can determine the presence, absence, or one or more characteristics of the target analyte. If the analyte is a polymer containing polymer units, the electrical measurement in the analysis can be processed to derive the estimated identity of the polymer units, count the polymer units, or determine the length of the polymer. Control experiments can be performed in the presence of different analytes or polymer units to determine how the analyte affects the electrical measurement as a basis for the analysis.

[0263] The analysis may be performed using any suitable known technique, including, for example, a technique using a Hidden Markov Model as described in International Publication WO-2013 / 041878 or International Publication WO-2015 / 140535; for example, a technique using machine learning as described in the literature [Boza et al., "DeepNano: Deep recurrent neural networks for base calling in MinION nanopore reads", PLoS ONE 12(6): e0178751, 5 June 2017]; for example, a technique using comparison of feature vectors as described in International Publication WO-2013 / 121224; or any other suitable technique.

[0264] These interactions may occur as the analyte moves, for example, by translocating through the pores. In such cases, electrical measurements can be performed as the analyte moves through the pores. This movement may occur while a potential difference is applied between the droplets, i.e., across the pores. The applied potential typically results in the formation of a complex between the pores and the polynucleotide-binding protein. The applied potential difference may be a voltage potential. Alternatively, the applied potential difference may be a chemical potential. An example of this is the use of a salt gradient across an amphiphilic layer. Salt gradients are disclosed in the literature [Holden et al., J Am Chem Soc. 2007 Jul 11;129(27):8650-5].

[0265] The target analyte may be a metal ion, inorganic salt, polymer, amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide, polynucleotide, dye, bleach, pharmaceutical, diagnostic agent, recreational drug, explosive, or environmental contaminant.

[0266] The analyte may be an amino acid, a peptide, a polypeptide, and / or a protein. The amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may contain amino acids synthesized or modified therein. Many different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are listed above. For the purposes of the present invention, it should be understood that the target analyte may be modified by any method available in the art.

[0267] The protein in the analyte may be an enzyme, antibody, hormone, growth factor, or growth regulatory protein, such as a cytokine. The cytokine may be selected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IL-13; interferons, preferably IL-γ; and other cytokines, such as TNF-α. The protein may be a bacterial protein, a fungal protein, a viral protein, or a parasite-derived protein.

[0268] The target analyte may be a nucleotide, an oligonucleotide, or a polynucleotide. Nucleotides and polynucleotides are discussed below. Oligonucleotides are typically short nucleotide polymers having 50 or fewer nucleotides, e.g., 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. Oligonucleotides may include any of the nucleotides discussed below, including abasic and modified nucleotides.

[0269] At least some of the polynucleotides can be double-stranded.

[0270] Polynucleotides can be nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotides may comprise a single strand of RNA hybridized to a single strand of DNA.

[0271] Polynucleotides can be of any length. For example, a polynucleotide can be at least 10, at least 50, at least 100, or at least 500 nucleotide pairs long. A polynucleotide can be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 nucleotide pairs long.

[0272] Any number of polynucleotides may be investigated. For example, the method of the present invention may relate to characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they may be different polynucleotides or two examples of the same polynucleotide.

[0273] Polynucleotides can occur naturally or be artificial.

[0274] If the analyte is a polynucleotide containing nucleotides and the estimated identity of the polymer unit is derived from electrophoretic measurements, strand characterization / sequencing or exonuclease characterization / sequencing may be applied.

[0275] In strand sequencing, polynucleotides can be translocated through nanopores with or against an applied potential. In such cases, electromeasures reveal one or more features of multiple nucleotides.

[0276] The droplet may contain a polymer binding moiety, such as an enzyme, to control the translocation of the polymer through the pore. The moiety may be a molecular motor that utilizes enzymatic activity, for example, if the moiety is an enzyme, or it may exist as a molecular brake.

[0277] When the polymer is a polynucleotide, many methods have been proposed to control the rate of translocation, including the use of polynucleotide binding molecules. Enzymes suitable for controlling the translocation rate of polynucleotides include, but are not limited to, polymerases, helicases, exonucleases, single-stranded and double-stranded binding proteins, and topoisomerases, such as girases. For other polymer types, moieties that interact with the corresponding polymer type may be used. Polymer interaction moieties are described in International Publication WO-2010 / 086603, International Publication WO-2012 / 107778, and the literature [Lieberman KR et al, J Am Chem Soc. It may be any moiety disclosed in [2010;132(50):17961-72)] and disclosed for voltage switching methods (Luan B et al., Phys Rev Lett. 2010;104(23):238103]).

[0278] Polymer binding moiety can be used in many ways to control polymer movement. The moiety can move the polymer through nanopores with or against the applied field. The moiety can be used as a molecular motor utilizing enzymatic activity, for example, if the moiety is an enzyme, or as a molecular brake. Translocation of the polymer can be controlled by a molecular ratchet that controls the movement of the polymer through the pores. The molecular ratchet can be a polymer binding protein. For polynucleotides, the polynucleotide binding protein is preferably a polynucleotide handling enzyme.

[0279] Preferred polynucleotide handling enzymes are polymerases, exonucleases, helicases, and topoisomerases, such as girases. The polynucleotide binding enzyme may be one of the types of polynucleotide handling enzymes described, for example, in WO-2015 / 140535 or WO-2010 / 086603.

[0280] In one embodiment, one or more membranes may be optional membranes having a plurality of pores inserted to provide a type of frit alternative for supplying a reagent. Such an embodiment may be used in a three-pore system, for example, comprising a droplet pair and a third droplet connected to the droplets of the droplet pair, wherein the interface between the third droplet and the droplets of the droplet pair comprises a plurality of pores. The third droplet is, for example, an electrochemical mediator, such as ferricyanide [Fe(CN)6] 3- / 2- It may include.

[0281] Different droplet interfaces may have different membrane-penetrating pores inserted thereon.

[0282] coupling

[0283] The analyte may contain an anchor for coupling the analyte to a membrane, or a tether for coupling the analyte to a pore. The membrane may be functionalized to facilitate the coupling of the analyte. The pore may be modified to facilitate the tethering of the analyte. Methods for coupling the analyte to a membrane known in the art may be used, for example, as described in International Publication WO-2012 / 164270 or International Publication WO-2015 / 150786. Methods for coupling the analyte to a pore known in the art may be used, for example, as described in International Publication WO-2012 / 164270 or International Application PCT / GB2017 / 053603.

[0284] Sample

[0285] A droplet (1) can be prepared from a sample. Such a sample may be known to contain an analyte or suspected to contain an analyte.

[0286] The sample may be a biological sample. The sample may be obtained from or extracted from any organism or microorganism.

[0287] The sample may be obtained from or extracted from any virus.

[0288] The sample is preferably a fluid sample. The sample typically contains the patient's body fluid. The sample may be urine, lymph, saliva, mucus, or amniotic fluid, but is preferably blood, plasma, or serum.

[0289] The sample may be of human origin, but alternatively, the sample may be from other mammals, for example, commercially raised animals, such as horses, cattle, sheep, fish, chickens, or pigs, or alternatively, from pets, such as cats or dogs. Alternatively, the sample may be of plant origin, for example, a sample obtained from commercial crops, such as cereals, legumes, fruits, or plants.

[0290] The sample may be a non-biological sample or may be derived from it. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water, such as drinking water, seawater, or river water, and reagents for experimental testing.

[0291] Examples

[0292] Examples of use of the device (1) performed are as follows.

[0293] The device (1) of the example is designed to perform DNA sample preparation and sequencing on a single portable platform. The main sequencing element is a protein nanopore embedded in a polymer membrane formed at the droplet interface (2) between two aqueous droplets (1) in the AM-EWOD device (34) of the type described above.

[0294] In this example, the liquid of the droplet (1) was an aqueous solution, the droplet contained an amphiphilic molecule, which is a triblock copolymer of the type described above, in the form of a vesicle, and the fluid medium (50) was a pure alkane hydrocarbon.

[0295] The embodiment used an AM-EWOD device (31) having an array (50) of driving electrodes (48) as shown in FIG. 17, which is an image also illustrating two droplets (1). FIG. 17a was taken at the end of the first step of the method described above, at which time the droplets (1) were in a state of close energy gain with a gap (3) between them. FIG. 17b was taken after the end of the second step when the droplets (1) had relaxed to form a droplet interface (2). Each driving electrode (48) in the array had dimensions of 200 x 200 μm, which was much smaller than any droplet (1) used.

[0296] In the AM-EWOD device (31), a layer (58) of conductive material is patterned as shown in FIG. 18, where the box (120) represents the area where the image of FIG. 17 was taken. FIG. 18 shows contact pads (121) labeled C1 to C15 at the upper edge. A sensor electrode (100) connected to C2 (grounded) labeled on the contact pad (121) and a sensor electrode (100) connected to C5 (recorded) labeled on the contact pad (121) were used for electrical recording from the droplet interface (2) in panel FIG. 17b.

[0297] By integrating a recording electrode into the AM-EWOD device (31), voltage application and current recording including a gap DNA sequencing signal were facilitated.

[0298] In the example, current recording was performed using a standard patch clamp amplifier on individual droplet interfaces (2). To perform multi-channel recording from one array of droplet interfaces (2) in parallel, a multi-channel recording system may be used. In order to record of sufficient quality (< 1 pA rms @ 5 KHz) for DNA sequencing, the system must be virtually free of electrical noise. Therefore, preferably, the device (1) operates in two mutually exclusive modes referred to as EWOD mode and recording mode.

[0299] In EWOD mode, all features in the layer (58) of the conductive material are connected to a control electronics (38) that supplies part of the voltage required for movement. Because the EWOD uses a high frequency, large AC voltage field, recording cannot occur while the EWOD field is on. Specifically, the EWOD field generates noise that obscures the DNA signal. Therefore, once the droplet (1) is positioned as desired, the control electronics (38) is unplugged, but an internal switching component may be used alternatively. Once the EWOD is unplugged, a multipole switch is activated.

[0300] The entire device (1) is confined in a Faraday cage during recording to prevent interference from ambient noise. Therefore, during the recording mode, the droplet is not held in place by any electrically induced force.

[0301] Using the method described above, using the AM-EWOD element (34), three systems were created, each consisting of two droplets (1) having a droplet interface (2) between the droplets, as shown in FIG. 19.

[0302] The formation of the droplet interface (2) was performed as follows.

[0303] It was possible to combine the two droplets (1) by simply manipulating them under the application of a driving signal to the driving electrode (48), but it was difficult because the droplets (1) tended to fuse.

[0304] Instead, the method described above was used. Specifically, in the first step, energy was applied to the droplets (1) to form a rectangular shape with an aspect ratio greater than 1.5. The long edges of these shapes were brought closer by 1 to 2 pixels and centered. An example of this step applied to three droplets (1) is shown on the left side of FIG. 20.

[0305] In the second step, the driving signal was switched. The droplets naturally relaxed back into a relaxed circular shape, reducing their surface area to volume ratio. Relaxation causes the surfaces of the droplets to face each other across the gap (3), making contact and forming a droplet interface (2) (this may be referred to as passive formation). Using this approach, a DIB can be created between two or more droplets. An example of this step applied to three droplets (1) is shown on the right side of FIG. 20.

[0306] This formation of the droplet interface (2) was a reversible process.

[0307] Although the control electronic device (38) generated noise that obscured the DNA sequencing signal, it is still possible to observe large current events, such as the insertion of a void into the droplet interface (2). As a demonstration, the device (1) was configured so that the AM-EWOD element (34) could receive power in a low-noise mode, and the droplet was positioned to form a droplet interface while recording an electric current.

[0308] FIG. 21 illustrates the electric current recorded in this way. Within this signal, it is possible to observe the formation, unzipping, re-formation, and void insertion of the droplet interface (2). Multiple cycles of void insertion and membrane separation and recombination are presented. Thick black arrows indicate void insertion. Record time bars indicate the recording mode, EWODon time bars indicate the EWOD mode, and driving time bars indicate droplet driving and shaping. Off-scale noise is observed during droplet driving.

[0309] Gap insertion was observed as a jump in current from 0 to about 200 pA at 300 mV. After gap insertion, the voltage was switched to zero, and the system was switched to EWOD mode. After the droplet (1) was separated, the droplet interface was reformed. Note that during this time, noise exceeded the scale of the current recording device.

[0310] After that, the system was switched back to recording mode and a voltage of 300 mV was applied. After observing another gap insertion, the above cycle was repeated one or more times for a total of three gap insertions and two separations of droplet interfaces (2). This demonstrates the ability of the AM-EWOD device (34) to repeatedly form droplet interfaces (2), insert gaps, separate droplets (1), and re-form droplet interfaces (2).

[0311] DNA detection and sequencing were performed as follows.

[0312] By placing an amphiphilic molecule in the droplet (1) rather than the fluid medium (50), it becomes possible to form an asymmetric membrane. For example, the DNA droplet may have a lower concentration of polymer vesicles compared to the opposite droplet, or the DNA droplet may have an entirely different polymer composition. This can provide flexibility in optimizing sample preparation, DIB formation, pore insertion, DNA sequencing, or further processes.

[0313] In one example, a short DNA strand (adapter) was detected by using an asymmetric pair of droplets (1) as shown in FIG. 22. FIG. 22 illustrates an example of a droplet interface (2) comprising a membrane of amphiphilic molecules having a concentration of less than 2 mg / mL in the DNA droplet (top) to aid in pore insertion. A higher polymer concentration in the opposite droplet (bottom) contributes to stability.

[0314] FIG. 23 illustrates an example of a characteristic adapter signal obtained from the droplet interface in FIG. 22. The current level denoted by pA indicates an open air gap (2) and an adapter-occupied air gap. In FIG. 23, the characteristic squiggle of the adapter blockage is an easily recognizable signal establishing the quality of the air gap and the overall system configuration.

[0315] By applying the same approach, a sequencing signal from single-stranded DNA can be obtained. The droplet interface (2) was created from a droplet (1) containing a 3.6 Kb single-stranded DNA sample, sequencing reagents and enzymes, polymer vesicles, mediator buffers, and nanopores. The counter droplet (1) contained vesicles of amphiphilic molecules and mediators + salts to osmotically balance the DNA droplet (1).

[0316] After observing single-pore insertion, the control electronic device (38) is unplugged, and the electrode is switched to recording mode. During sequencing, the DNA strand is threaded into the pore and then pulled through by the applied voltage. The threading speed is controlled by an attached enzyme, that is, driven by ATP turnover within the droplet.

[0317] The DNA used in this experiment was of standard 3.6 Kb length with a known sequence, so each strand was expected to thread through the pores for a similar amount of time. Figure 24 illustrates an example of an electrical measurement performed, representing a DNA threading event at 180 mV. Note that the current interruption lasted from 15.1 to 18.3 seconds, which corresponds to approximately 200 bases per second. This is the expected translocation rate for the nanopores operating under experimental conditions. Since each DNA strand in the control sample is identical, the tangled sequences from each translocation event must be identical.

[0318] Figure 25 is a plot of expanded current traces for six translocation events, each showing a characteristic "α-basic" peak followed by a sequencing signal. Note that all traces have the same profile. The approximate alignment of these current traces for the six translocation events indicates that the signal patterns are identical for each DNA strand.

Claims

Claim 1 An electro-wetting device for performing measurements across a droplet interface, wherein the electro-wetting device comprises: an array of actuation electrodes and a first substrate supporting an insulating layer covering the actuation electrodes and having a hydrophobic surface; and a second substrate supporting at least one set of at least two sensor electrodes, wherein the electro-wetting device is configured to receive a fluid medium disposed between the first substrate and the second substrate and one or more droplets of a liquid suspended in the fluid medium, the droplets being disposed on the hydrophobic surface, and the actuation electrodes being configured to receive actuation signals for electro-wetting, moving, and / or deforming the shape of the one or more droplets to form at least one system of droplets having at least one droplet interface between the droplets, and the at least one set of sensor electrodes being arranged on the second substrate to make electrical contact with each droplet in the at least one system of droplets. Claim 2 An electro-wetting element according to claim 1, wherein the second substrate is disposed in a plane parallel to the plane of the first substrate and is spaced apart from the first substrate. Claim 3 An electro-wetting device according to claim 1 or 2, further comprising a control system connected to the driving electrode and configured to apply driving signals to the driving electrode to manipulate the droplet on the hydrophobic surface. Claim 4 In paragraph 3, the electro-wetting element is configured such that the control system applies driving signals to the driving electrodes to form at least one system of the droplet that is in electrical contact with at least one set of the sensor electrodes. Claim 5 In paragraph 4, the control system is an electro-wetting element configured to apply a reference signal to the sensor electrodes while applying driving signals to the driving electrodes. Claim 6 In claim 5, the control system is an electro-wetting element configured to apply driving signals to the driving electrodes selected to form a droplet interface at the interface between two droplets. Claim 7 An electro-wetting element according to claim 5, wherein the second substrate further supports at least one additional electrode, and the control system is connected to the additional electrode and configured to apply a reference signal to the additional electrode while applying driving signals to the driving electrodes. Claim 8 In claim 7, the additional electrode is an electro-wetting device that is deposited on the surface of the second substrate facing the first substrate. Claim 9 In claim 8, the additional electrode is an electro-wetting element extending around the sensor electrodes. Claim 10 In claim 5, the electro-wetting element further comprises a sensor system connected to the sensor electrode, wherein the sensor system is configured to perform electrical measurements between sensor electrodes electrically connected to each droplet forming a droplet interface. Claim 11 In paragraph 10, the above electrical measurement is an electro-wetting device including an impedance measurement. Claim 12 In claim 10, the sensor system is an electro-wetting device configured to perform electrical measurements between each sensor electrode that comes into contact between each droplet across a droplet interface comprising a membrane of amphiphathic molecules having an internally inserted transmembrane pore. Claim 13 In claim 12, the electro-wetting device, wherein the electrical measurements are measurements of ion flow between droplets through a transmembrane pore. Claim 14 In claim 12, the sensor system is an electro-wetting element configured to perform the electrical measurements while applying a potential difference between each pair of sensor electrodes. Claim 15 In claim 12, the sensor system is an electro-wetting element configured to perform electrical measurements dependent on the analyte interacting with the membrane-penetrating pore. Claim 16 In claim 15, the electro-wetting device further comprises an analysis system configured to process the electrical measurements in order to analyze the sample to be analyzed. Claim 17 In paragraph 16, the analyte is a polymer comprising polymer units, and the analysis system is configured to process the electrical measurements to derive the estimated identity of the polymer units of the polymer, an electro-wetting device. Claim 18 In claim 1, the second substrate is an electro-wetting element that supports a plurality of sets of at least two sensor electrodes. Claim 19 An electro-wetting device according to claim 1, wherein the insulating layer comprises a layer of an electrical insulating material coated with a hydrophobic material forming the hydrophobic surface. Claim 20 An electro-wetting device according to claim 1, wherein the sensor electrodes are deposited on the surface of the second substrate facing the first substrate. Claim 21 An electro-wetting device according to claim 20, further comprising a conductive track deposited on the surface of the second substrate and connected to the sensor electrode to provide an electrical connection to the sensor system. Claim 22 The electro-wetting element according to claim 1, wherein the second substrate is coated with a hydrophobic material forming an additional hydrophobic surface opposite to the hydrophobic surface of the insulator layer, and the electro-wetting element is provided to receive a fluid medium and droplets disposed on the additional hydrophobic surface of the hydrophobic material coating the second substrate as well as the hydrophobic surface of the insulator layer; and the hydrophobic material coating the second substrate has apertures that expose at least some of the sensor electrodes. Claim 23 In claim 21, the second substrate is an electro-wetting device that additionally supports at least one additional electrode. Claim 24 In paragraph 23, the additional electrode is an electro-wetting device that is deposited on the surface of the second substrate facing the first substrate. Claim 25 In paragraph 24, the additional electrode is an electro-wetting element extending around the sensor electrodes. Claim 26 An electro-wetting element according to claim 1, further comprising two droplets including the fluid medium and the liquid within the fluid medium, which are accommodated in the electro-wetting element. Claim 27 An electro-wetting device according to claim 26, wherein one of the liquid and the fluid medium is polar and the other of the liquid and the fluid medium is non-polar, the droplets further comprise amphiphilic molecules at the interface between the liquid of the droplets and the fluid medium, and the droplet interfaces comprise a film of amphiphilic molecules. Claim 28 In paragraph 27, the above liquid is polar and the above fluid medium is nonpolar, an electro-wetting element. Claim 29 In paragraph 26, the above fluid medium is a liquid medium, an electro-wetting element. Claim 30 In claim 26, an electro-wetting device comprising at least one of the droplets having a membrane-penetrating pore capable of being inserted into a membrane of amphiphilic molecules. Claim 31 In claim 30, an electro-wetting device comprising at least one of the droplets comprising an analyte capable of interacting with the membrane-penetrating pore.

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