Improvements in devices and methods for facilitating the handling of microdroplets

Abstract

A device for manipulating hundreds or thousands of microdroplets into an array using an EWOD or oEWOD is provided. The device includes: i) a chip including a first region for receiving and manipulating microdroplets, a second region including the array, and a plurality of electrowetting paths leading to the array; ii) a microdroplet source configured to provide microdroplets of a predetermined target diameter; iii) a channel configured to provide fluid communication between the microdroplet source and the first region of the chip; and iv) a pressure source configured to move the microdroplets between the microdroplet source and the first region of the chip. The plurality of electrowetting paths on the chip are separated by a center-to-center distance at least twice the predetermined target diameter of the microdroplets from the microdroplet source. Furthermore, a controller is configured to enable synchronous movement of the microdroplets within the electrowetting paths by application of an EWOD or oEWOD force.

Description

Technical Field

[0001] The present invention relates to a device and method for facilitating the manipulation of microdroplets, and more particularly, to a device and method for loading one or more microdroplets into a microfluidic chip.

Background Art

[0002] Electrowetting-on-dielectric (EWOD) is well known as an effect in which when an electric field is applied between a liquid and a substrate, the liquid becomes more wettable on the surface than in its natural state. By utilizing the effect of electrowetting, microdroplets can be manipulated. The effect of electrowetting can be used for the manipulation of microdroplets (for example, control of movement, confluence, splitting, shape change, etc. of microdroplets) by applying a spatially varying series of electric fields on the substrate and increasing the wettability of the surface following the series of spatial variations. Droplets operated by a device using electrowetting are usually sandwiched between two parallel plates and actuated by digital electrodes. The size of the pixelated electrodes limits the minimum droplet size that can be operated, as well as the speed and scale at which droplets can be processed in parallel.

[0003] A variation of this method provides the driving force for devices for manipulating microdroplets using light-mediated electrowetting forces, known in the art as optoelectrowetting. In this optoelectrowetting (oEWOD) device, microdroplets are repositioned through a microfluidic space defined by containment walls, for example, a pair of parallel plates with a microfluidic space in between. At least one of the containment walls includes what will be hereafter referred to as "virtual" electrowetting electrode positions, generated by selectively illuminating an embedded semiconductor layer with light. By selectively illuminating the layer with light from a separated light source controlled by an optical assembly, a virtual path for the virtual electrowetting electrode positions is transiently generated, along which the microdroplets can move. This eliminates the need for conductive cells and generates temporary droplet receiving positions on a homogeneous dielectric surface by selectively and variably illuminating points on the photoconductive layer, for example, using a pixelated light source, rather than permanent droplet receiving positions. This makes it possible to generate highly localized electrowetting fields at any location on the dielectric layer, where induced capillary forces can move microdroplets on the surface, and to arbitrarily associate them with a microfluidic flow in any direction of the carrier medium in which the microdroplets are dispersed, for example by emulsification.

[0004] One example of EWOD and oEWOD device applications is in the pharmaceutical industry, specifically in cell line development and antibody development. In these fields, it's necessary to perform initial screening on a large number of biological agents (up to several million) to reduce the number of drugs to a more manageable number (several thousand). To achieve an efficient workflow, this initial screening needs to be performed redundantly on a large number of biological agents.

[0005] Therefore, a key aspect of EWOD or oEWOD devices intended for use in these fields is the ability to process large quantities of droplets—hundreds, thousands, or even millions—at once. Existing EWOD and oEWOD devices use optical microscopes to handle samples, which imposes practical limitations on the number of droplets that can be processed in parallel within a single field of view. Existing devices are limited to processing several thousand droplets at a time.

[0006] Essential features of EWOD or oEWOD devices capable of processing and manipulating millions of droplets include numerous optical manipulation spots, scale-up chips, and the ability to quickly and reliably load millions of droplets into the device.

[0007] Existing methods for loading droplets into EWOD and oEWOD devices depend on manual human intervention suitable for delivering small droplets to the tip, the extraction of droplets from the flow at the edge of the tip which suffers from performance issues due to inaccurate control of droplet velocity, or the design of a device that loads droplets in bulk into a holding pen before ejection. In the former, the maximum flow rate is limited by the maximum flow rate of the droplet EWOD or oEWOD, and a large area of ​​the device is wasted. The latter is inherently prone to problems due to processing switchover time, as it is essentially batch processing.

[0008] Therefore, there is a need to provide a device and method for rapidly and efficiently loading multiple microdroplets onto a chip. Furthermore, there is a need to load the droplets onto the chip in a way that allows for easy and simple manipulation by EWOD or oEWOD force.

[0009] Furthermore, a significant proportion of droplets loaded into an EWOD or oEWOD device may be unsuitable for analysis. For example, droplets may be of an undesirable size that makes them difficult to select and manipulate by the EWOD or oEWOD force. To maximize the space capacity of desirable droplets within the device, it is important to remove undesirable droplets as early as possible in the loading process so that unwanted droplets do not occupy space within the chip. Also, the contents of the droplets may be undesirable. For example, in an analysis starting with one cell per droplet, empty droplets or droplets containing multiple cells are undesirable. By removing droplets that do not meet the criteria for acceptable content, the yield of useful droplets retained for analysis can be increased.

[0010] Therefore, there is a need to provide devices and methods that can efficiently control and manipulate millions of microdroplets by EWOD or oEWOD force while optimizing the use of space on the chip. Furthermore, there is a need to provide devices, apparatus and / or methods that can quickly and efficiently identify and separate undesirable droplets from millions of droplets loaded onto the chip. It is desirable that the device be able to handle the removal of undesirable droplets and maintain a constant yield of droplets in the array even when a large number of undesirable droplets are removed from the device. Moreover, it is highly desirable to provide a fast and efficient apparatus for removing undesirable droplets from the chip at the beginning of the droplet manipulation process.

[0011] This invention arose from this technical background. [Overview of the project]

[0012] According to a first aspect of the present invention, a device is provided comprising: i) a tip including a first region for manipulating a plurality of microdroplets; ii) a microdroplet source for supplying the microdroplets; iii) a channel having a distal end extending in a first direction within the tip and a proximal end in fluid communication with the microdroplet source; and iv) a pressure source for moving the microdroplets from the microdroplet source along the channel to the first region of the tip, wherein the pressure source is configured to enable the movement of the microdroplets from the microdroplet source to the proximal end of the channel at a first velocity, and the distal end of the channel is grooved or blunted to allow the microdroplets to move from the distal end of the channel to the first region of the tip at a velocity lower than the first velocity.

[0013] In one embodiment, the microdroplet source may be a reservoir for holding microdroplets. In another embodiment, the microdroplet source may be a droplet generator, such as an emulsifier device, for generating droplets.

[0014] In one embodiment, the pressure source for moving the microdroplets is a pump. The pump may be configured to apply negative pressure at the outlet and / or positive pressure at the microdroplet source in order to move the microdroplets.

[0015] In one embodiment, the device comprises i) a tip including a first region for manipulating one or more microdroplets; ii) a reservoir for holding one or more microdroplets; iii) a channel extending in a first direction within the tip and in fluid communication with the reservoir; iv) means for moving one or more microdroplets between the reservoir and the first region of the tip; and v) at least one outlet provided in the tip, wherein the channel, the first region and the at least one outlet are configured such that one or more microdroplets flow from the reservoir to the first region at a first velocity, and that the one or more microdroplets move within the first region at a velocity lower than the first velocity.

[0016] In one embodiment, a droplet generator is provided for generating one or more microdroplets, and the droplet generator may be in fluid communication with the chip. The droplet generator may be an emulsifier device for generating droplets. In one embodiment, the emulsifier device may be a step emulsifier device. This may be advantageous because the step emulsifier device can operate continuously to generate a large number of droplets. Therefore, preparing a droplet generator such as an emulsifier device is particularly useful for generating a large number of microdroplets over a long period of time. The droplet generator may be in fluid communication with the chip via a channel extending in a first direction into the chip. Microdroplets generated by the droplet generator can be moved to a first area of ​​the chip by the operation of a pressure source. Using a droplet generator is advantageous because it eliminates the need to pipette the droplets once they have been generated. The droplet generator may be provided within the apparatus of the present invention. Those skilled in the art will understand that any form of droplet generator can be used. Furthermore, those skilled in the art will understand that droplets can be generated using any type of emulsifying device and then transported to a chip.

[0017] Alternatively, or in addition to, a droplet generator, the apparatus of the present invention may be provided with a reservoir for holding one or more microdroplets.

[0018] A device configured to allow one or more microdroplets to move in a first region at a speed lower than a first velocity requires that the droplets be forced to stop effectively once they enter the first region of the device. This is important for efficiently removing the droplets from the flow and controlling them within the device using an EWOD or oEWOD. Devices according to any embodiment of the present invention can be used to process large volumes of microdroplets ranging from hundreds to millions.

[0019] The devices provided herein may further comprise two or more outlets provided on the chip. In some embodiments, at least one outlet is located on either side of the channel. Providing outlets on the chip allows for a directional flow from inlet to outlet. Outlets are an option when microdroplets are loaded onto the chip and then remain there without a specific discharge scheme. In some embodiments, an outlet is provided for initiation purposes but is closed during the subsequent loading process and remains closed throughout subsequent operations.

[0020] In one embodiment, the channel of the device provided herein comprises a proximal end through which one or more microdroplets move from a droplet source into the channel, and a distal end through which one or more microdroplets move from the channel into a first region of the chip.

[0021] In some embodiments, the distal end of the channel may be blunted or grooved to create a section with a different cross-sectional area from the channel to alter the velocity of microdroplets passing through it; i.e., the final section of the channel is tapered inward or outward, respectively. In some embodiments, the flare angle of the blunted or grooved end is 0 to < 90°. The flare angle of the blunted or grooved end may be 0, 10, 20, 30, 40, 50, 60, 70, or 80° or more. In some embodiments, the flare angle of the blunted or grooved end may be less than 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5°. Preferably, the angle may be 45° or 75°. In some embodiments, the channel wall at the distal end of the channel may be rounded or square.

[0022] To reduce the flow velocity in the first region, the distal end of the channel extending into the first region of the device may be blunt-ended or grooved. The change in shape where the blunt-ended or grooved channel end meets the first region facilitates a rapid decrease in flow velocity, causing the droplet to effectively stop upon reaching the distal end of the channel. This enables droplet loading at maximum speed without compromising efficient EWOD or oEWOD operation and droplet control. Since the EWOD or oEWOD force must overcome the flow, high flow velocity is detrimental to the operation of the EWOD or oEWOD. Therefore, reducing the droplet velocity enables effective EWOD or oEWOD operation and also improves space efficiency within the device. It will be understood by those skilled in the art that the distal end of the channel can be any suitable shape to facilitate a rapid decrease in flow velocity.

[0023] In one embodiment, the channel of the device provided herein extends into the chip for a distance of 1000 μm or more. This ensures that the distal end of the channel is located at a sufficient distance from the outlet to facilitate a rapid decrease in flow rate.

[0024] In one embodiment, the portion of the channel protruding into a first region of the device may have a length between 1,000 and 20,000 μm. In one embodiment, the protruding length of the channel may be greater than 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 2,200, 2,400, 2,600, 2,800, 3,000, 3,200, 3,400, 3,600, 3,800, 4,000, 4,200, 4,400, 4,600, 4,800, 5,000, 5,200, or 5,400 μm. In some embodiments, the channel projection length may be less than 5500, 5400, 5200, 5000, 4800, 4600, 4400, 4200, 4000, 3800, 3600, 3400, 3200, 3000, 2800, 2600, 2400, 2200, 2000, 1800, 1600, 1400, or 1200 μm. The minimum channel length may be 250 μm. The minimum channel length may be necessary to form a low-velocity region at the distal end of the channel and to prevent a substantial component of the flow that moves directly between the channel end and the outlet, which would prevent droplets from effectively stopping when they reach the distal end of the channel. In some embodiments, a minimum fan length is used to create a backflow in the direction of flow, which essentially results in a decrease in microdroplet velocity at the channel end.

[0025] In one embodiment, the channel of the device provided herein has a distance of 1500 μm or more between the channel and at least one outlet. In one embodiment, the channel of the device provided herein has a distance of 3600 to 5600 μm between the channel and at least one outlet. In one embodiment, the distance between the channel and at least one outlet may be 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, or up to 11200 μm or more. In one embodiment, the distance between the channel and at least one outlet may be less than 5600, 5500, 5400, 5300, 5200, 5100, 5000, 4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100, 4000, 3900, 3800, or 3700 μm. Sufficient separation of the distal end of the channel and the outlet is necessary to prevent a substantial component of the flow moving directly between the distal end of the channel and the outlet from hindering droplets at the distal end of the channel from achieving a sharp decrease in flow rate.

[0026] In some embodiments, the channels of the devices provided herein may be tapered. In other embodiments, the width of the channels is substantially the same along their entire length. In some embodiments, the width of the channels is between 300 μm and 25 mm. In some embodiments, the width of the channels can be greater than 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680 μm. In some embodiments, the width of the channels can be less than 700, 680, 660, 640, 620, 600, 580, 560, 540, 520, 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 80, 60 or 40 μm. In some embodiments, the width of the channels can be up to several millimeters. The minimum channel width is equal to the minimum droplet diameter so as not to compress or distort the droplets when loading them into the channels. The maximum length of the channels is limited by the exit position and the space within the chip.

[0027] In some embodiments of the devices provided herein, the first speed corresponds to a flow rate between 0.1 and 100 μL / min. In some embodiments, the flow rate can be greater than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 μL / min. In some embodiments, the flow rate can be less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1. 0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2 μL / min. In other embodiments, the first speed corresponds to a flow rate between 0.1 and 0.4 μL / min. In some embodiments, the flow rate can be greater than 0.10, 0.15, 0.20, or 0.25 μL / min. In some embodiments, the flow rate can be less than 0.40, 0.35, 0.30, 0.25, 0.20, or 0.15 μL / min.

[0028] In certain embodiments of the devices provided herein, the velocity of the microdroplets in the first region can be from 25 to 5000 μm / second. In certain embodiments, the velocity of the microdroplets can be greater than 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900 or 1950 μm / second. In certain embodiments, the velocity of the microdroplets can be less than 2000, 1950, 1900, 1850, 1800, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 μm / second. Thereby, the EWOD or oEWOD force can effectively manipulate the droplets, facilitating the self - organization of the droplets by EWOD or oEWOD control and subsequent formation of an ordered array.

[0029] In certain embodiments of the devices provided herein, the surface area of the first region can be greater than the internal surface area of the channel.

[0030] In certain embodiments of the devices provided herein, the means for moving one or more microdroplets can be a pressure source such as a pump. In certain embodiments, the pump can be configured to apply negative pressure at the outlet and / or positive pressure at the reservoir to move one or more microdroplets. In certain embodiments, the pump is configured to apply negative pressure at the outlet to move one or more microdroplets.

[0031] In some embodiments of the devices provided herein, the average spherical microdroplet diameter may be 20 to 200 μm. In some embodiments, the average microdroplet diameter may be greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 μm. In some embodiments, the average microdroplet diameter may be less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 μm. In other embodiments, the average microdroplet diameter is 50 to 100 μm. In some embodiments, the average microdroplet diameter can be 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 μm or larger. In some embodiments, the average microdroplet diameter may be less than 100, 95, 90, 85, 80, 75, 70, 65, 60, or 55 μm.

[0032] In some embodiments of the devices provided herein, the chip may be an EWOD chip. In some embodiments, the chip is an oEWOD chip.

[0033] In one embodiment, the chip includes a second region containing a desired array position, through a plurality of electrowetting paths formed by the application of transient EWOD or oEWOD forces at positions along the paths, which move microdroplets from a first region to a second region of the chip. The plurality of electrowetting paths formed by transient EWOD or oEWOD forces allow for the continuous movement of microdroplets and can facilitate parallel loading and manipulation of droplets within the chip.

[0034] In one embodiment, the device provided herein provides one or more electrowetting paths, and within each path, each microdroplet All other microdroplets The system may further include a microprocessor configured to synchronize movements relative to it.

[0035] In one embodiment of the device provided herein, the microdroplets in the first region are random, while the microdroplets in the second region are aligned. In terms of aligning the microdroplets, multiple microdroplets may be arranged in a series of parallel rows.

[0036] In some embodiments, optical spectroscopy, such as light or fluorescence spectroscopy, can be used to detect one or more microdroplets. In some embodiments, the detector may be configured to detect the fluorescence of one or more microdroplets. In some embodiments, the detector may be a fluorescence detector.

[0037] In a further embodiment of the present invention, a method is provided for loading microdroplets onto a chip for operation, the method comprising: a) preparing the device described herein; b) moving one or more microdroplets from a reservoir to a first region via a channel extending in a first direction; and c) manipulating the microdroplets in the first region, wherein the one or more microdroplets flow from the reservoir to the first region at a first velocity, and the one or more microdroplets move in the first region at a velocity lower than the first velocity.

[0038] In one embodiment, the microdroplet loading rate onto the chip may exceed 35 droplets / second, and even reach 70 droplets / second. This allows for efficient full loading of the device, for example, loading millions of microdroplets into the device in less than 8 hours, or even as little as 4 hours in some cases.

[0039] According to another aspect of the present invention, a device is provided for manipulating hundreds or thousands of microdroplets into an array using EWOD or oEWOD, the device comprising: a chip having i) a first region for receiving and manipulating microdroplets, an array, and a second region including a plurality of electrowetting paths leading to the array; ii) a microdroplet source configured to supply microdroplets of a predetermined target diameter; iii) a channel configured to provide fluid communication between the microdroplet source and the first region of the chip; and iv) a pressure source configured to move microdroplets between the microdroplet source and the first region of the chip, wherein the plurality of electrowetting paths on the chip are center-to-center at a distance of at least twice the predetermined target diameter of the microdroplets from the microdroplet source, and a controller is configured to enable synchronous movement of microdroplets in the electrowetting paths by application of EWOD or oEWOD force.

[0040] The pressure source may be configured to apply positive or negative pressure to push or pull microdroplets from a microdroplet source into a first region of the tip.

[0041] Providing an electrowetting path that is at least twice the average microdroplet diameter may be advantageous in order to allow a single microdroplet to pass between two other microdroplets. This is necessary to allow droplets not controlled by EWOD or oEWOD forces to fall between microdroplets controlled by EWOD or oEWOD forces. Thus, this is necessary to obtain a sieving effect and for droplets to self-organize. The sieving effect is a remarkable technical effect resulting from the present invention. The sieving effect can be optimized by moving droplets at the maximum speed possible with the available EWOD forces, thereby retaining only droplets with optimal sprite-droplet overlap, so that each sprite controls a single droplet, thus promoting self-organization. This process can be further optimized in EWOD by lowering the droplet holding potential in this section of the path, and in oEWOD by lowering the incident electromagnetic radiation of the sprites. When using EWOD, this reduction in retention affinity can be achieved in various ways, including, but is not limited to, changing the shape of the ether electrode, reducing the applied electric field, and shifting the AC frequency. This reduction in retention affinity is particularly useful when applied to the self-assembly region because it limits the time the droplet travels at near-maximum speed to this region only, allowing it to comfortably travel at or below maximum speed without changing speed in the rest of the path. This is crucial for maximizing droplet retention and loading rates. The reason why a decrease in droplet speed after self-assembly is undesirable is that it can lead to narrower spacing between droplets, loss of control, collisions between droplets, or changes in droplet retention potential. In the case of oEWOD, the illumination intensity in the self-assembly region can be between 0.01 and 0.99 of the intensity used in the rest of the path. In very high-quality devices with a low probability of accidental droplet loss, higher initial light intensities, such as 0.8, between 0.75 and 0.99, can be used. This allows for the use of higher loading speeds.In low-quality devices where droplet loss is more likely, it may be necessary to use low light intensity ratios such as 0.01–0.5, which further minimizes the risk of droplet loss but compromises the maximum loading speed. In other devices, it may be optimal to use light intensity ratios between 0.5 and 0.75. Furthermore, or alternatively, spacing between electrowetting paths can help reduce or minimize the risk of droplets from different electrowetting paths coming into contact with each other. Spacing between electrowetting paths can allow droplets to move efficiently and continuously along the path until they are sorted and selected by the user or an automated software controller for operation. This operation is particularly effective when dealing with a large number of microdroplets in a series of multiple electrowetting paths, facilitating the efficient organization of droplets from cluttered droplets.

[0042] Furthermore, electrowetting paths can be arranged within the microfluidic chip region to maximize the available space or volume within the region for droplet manipulation and / or control. Electrowetting paths can be arranged in parallel, or they can be actuated by a controller to rotate within the chip. Electrowetting paths can be arranged in any suitable manner to utilize the maximum available space within the microfluidic chip, which may be particularly useful when utilizing chips with additional internal structures such as supports.

[0043] In one embodiment, droplets can be moved between electrowetting paths to efficiently redistribute them before the final array, which is particularly useful when droplets arrive in the first region in a consistently non-uniform manner.

[0044] In one embodiment, microdroplets may be manipulated using oEWOD. oEWOD manipulation of microdroplets can be performed continuously, maximizing efficiency while eliminating the need for isolation or holding pens.

[0045] A chip according to any one aspect of the present invention provided herein further comprises a first region for receiving and manipulating microdroplets and a second region including an array, wherein a plurality of electrowetting paths facilitate flow communication between the first and second regions.

[0046] In one embodiment of the chip provided herein, the electrowetting path is created by one or more sequences of moving sprite patterns.

[0047] A sprite pattern is an array of one or more individual sprites, where a sprite is a highly localized electric field formed by photoexciting the photoconductive layer of the chip.

[0048] In one embodiment of the chip, the number of sprites in a given path can be any appropriate number and may change over time as sprites can be added to or removed from the electrowetting path. This allows for the continuous growth of the sprite pattern.

[0049] In some embodiments of the chip provided herein, each individual sprite can control a single droplet. This ensures precise control of microdroplets and their self-organization into an array.

[0050] In some embodiments of the chip provided herein, the velocity of microdroplets in the electrowetting path may be 25 to 5000 μm / second. In some embodiments, the velocity of microdroplets in the electrowetting path may be 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250 , 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900 or 1950, 2000, 2200, 2500, 2700, 3000, 3200, 3500, 3700, 4000, 4200, 4500, 4700 μm / sec. In one embodiment, the velocity of microdroplets in the electrowetting path is 5000, 4700, 4500, 4200, 4000, 3700, 3500, 3200 The speeds may be 3000, 2700, 2500, 2200, 2000, 1950, 1900, 1850, 1800, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, or less than 50 μm / sec. This enables effective manipulation of droplets by EWOD or oEWOD forces and promotes the self-organization of droplets into an aligned array.

[0051] Aligned arrays can be particularly useful in the area of ​​a microfluidic chip for droplet manipulation and / or control, especially in narrow, compact spaces, as they allow the user to maximize available space and / or capacity. This aligned approach allows for the efficient organization of future operations such as droplet merging and splitting.

[0052] In one embodiment of the apparatus described herein, the spacing between electrowetting paths may be at least twice the average droplet diameter.

[0053] In one embodiment, the center-to-center distance of the electrowetting path may be at least the average droplet diameter.

[0054] Providing electrowetting paths that are at least twice the average microdroplet diameter may be advantageous in order to allow a single microdroplet to pass between two other microdroplets. This is necessary to allow droplets not controlled by EWOD or oEWOD forces to fall between microdroplets controlled by EWOD or oEWOD forces. Thus, it is necessary for the sieving effect and droplet self-organization. The sieving effect is a remarkable technical effect resulting from the present invention. Additionally or alternatively, spacing between electrowetting paths may help reduce or minimize the risk of droplets from different electrowetting paths coming into contact with each other. Spacing between electrowetting paths allows droplets to move efficiently and continuously along the path until they are sorted and selected by the user or an automated software controller for operation. This operation is particularly effective when dealing with a large number of microdroplets in a series of multiple electrowetting paths, facilitating the efficient sorting of droplets from clutter.

[0055] In one embodiment, droplets can be moved between electrowetting paths to efficiently redistribute them before the final array, which is particularly useful when droplets arrive in the first region in a consistently non-uniform manner.

[0056] In some embodiments of the chip provided herein, the spacing between electrowetting paths is 2 to 4 times the average microdroplet diameter. In some embodiments of the chip provided herein, the spacing between electrowetting paths may be 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 times, 3, 3.2, 3.4 or 3.6 times or more of the average microdroplet diameter. In some embodiments of the chip provided herein, the spacing between electrowetting paths may be less than 4, 3.8, 3.6, 3.4, 3.2, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2 or 2.1 times the average microdroplet diameter. The preferred distance between electrowetting paths is 2.5 times the average microdroplet diameter, which prevents droplets from spontaneously moving between electrowetting paths without controller intervention.

[0057] In some embodiments of the tips provided herein, the average spherical microdroplet diameter may be 20 to 200 μm. In some embodiments, the average microdroplet diameter may be 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 μm or more. In some embodiments, the average microdroplet diameter may be less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30.

[0058] In another embodiment of the device provided herein, the average microdroplet diameter is 50–100 μm. In some embodiments, the average microdroplet diameter may be 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 μm or greater. In some embodiments, the average microdroplet diameter may be less than 100, 95, 90, 85, 80, 75, 70, 65, 60, or 55 μm. In this context, the term “microdroplet diameter” refers to the effective spherical diameter of an unconstrained microdroplet. This is different from the apparent “diameter” of a microdroplet after deformation during loading into the device.

[0059] In one embodiment, the center-to-center distance of the electrowetting path is at least 100 μm for microdroplets with a diameter of 100 μm. This prevents droplet movement between electrowetting paths unless activated by a controller.

[0060] In some embodiments of the chip provided herein, the number of electrowetting paths present is between 2 and 250. In some embodiments, the number of electrowetting paths present can be between 40 and 180, or between 200 and 250. In some embodiments, the number of electrowetting paths present may be more than 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 210, 220, 230, or 240. In some embodiments, the number of electrowetting pathways present may be 250, 240, 230, 220, 210, 200, 180, 160, 140, 120, 100, 80, 60, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, or less than 4. In some embodiments of the chip provided herein, the number of electrowetting pathways present is 3 to 10. In some embodiments, the number of electrowetting pathways present may be more than 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the number of electrowetting pathways present may be 10, 9, 8, 7, 6, 5, or less than 4. In another example, approximately 180 electrowetting pathways can be provided to accommodate a droplet size of 50 μm in diameter.

[0061] In some embodiments of the chip provided herein, two or more electrowetting paths may propagate from a first region at different angles.

[0062] In some embodiments of the chip provided herein, two or more electrowetting paths may propagate from a first region at substantially the same angle.

[0063] In some embodiments of the chip provided herein, one or more electrowetting paths may be divided to form two or more electrowetting paths. In some embodiments, one or more electrowetting paths may be combined to form at least one additional electrowetting path. This facilitates droplet manipulation and allows for the separation of undesirable droplets from the rest of the droplet array.

[0064] In one embodiment of the chip provided herein, the electrowetting path is such that each microdroplet in the path All other microdroplets It is created by a controller configured to synchronize its movement with that of the microdroplets. The controller may be a software controller. This allows one or more microdroplets to be moved by the operation of the controller without disturbing other microdroplets in the electrowetting path.

[0065] According to another aspect of the present invention, an apparatus is provided for manipulating one or more microdroplets into an array using an EWOD or oEWOD, the apparatus comprising: a chip for manipulating microdroplets, having a plurality of electrowetting paths leading to an array and one or more waste electrowetting paths leading to a waste outlet; a detector configured to acquire a measurement dataset related to the distinct characteristics of a detected microdroplet in order to detect one or more microdroplets having distinct characteristics; a storage module configured to store and hold a stored dataset related to the characteristics measured by the detector; and a controller configured to receive the stored dataset and acquired measurement dataset from the storage module and to determine whether the measurement dataset is related to a desired or undesirable characteristic, wherein the controller is configured to select one or more microdroplets having a measurement dataset related to an undesirable characteristic and to move one or more selected microdroplets to a waste electrowetting path. Furthermore, the controller may be configured to select one or more microdroplets having a measurement dataset related to a desired characteristic and to move one or more selected microdroplets to an electrowetting path leading to an array.

[0066] The electrowetting paths described herein are temporarily generated by applying a series of selectively and spatially varying electric fields to the substrate of the EWOD device. Alternatively, the electrowetting paths described herein are temporarily generated by applying a series of selectively and spatially varying point illuminations to the photoconductive layer of the EWOD device.

[0067] A microdroplet may be considered undesirable if its measurements relate to a specific characteristic that is equal to, above, or below one or more stored thresholds set by the user. Alternatively, the desirability of a droplet may be determined from a combination of characteristics, a time-varying analysis of these characteristics, or the average measurement of these characteristics.

[0068] A waste electrowetting path is an electrowetting path extending from a first region of the device to an exit within the chip. In some configurations, one or more waste electrowetting paths may exit from one or more electrowetting paths. The waste electrowetting path facilitates the removal of undesirable droplets from the electrowetting paths and removes undesirable droplets from the chip. In some embodiments, the controller is configured to select one or more microdroplets having a measurement data set associated with undesirable characteristics and to move one or more selected microdroplets to one or more waste electrowetting paths. In some embodiments, the controller may be configured to select one or more microdroplets having a measurement data set associated with multiple undesirable characteristics. For example, the controller may be configured to select one or more microdroplets that are determined to be smaller than normal in size and / or empty.

[0069] In one embodiment, the controller may be a software controller. In another embodiment, the controller may be a microcontroller.

[0070] The distinct characteristics of the detected microdroplets described herein may include, but are not limited to, the number of objects within them, the shape of the droplets, the size of the droplets, and the intensity of fluorescence or light transmitted through the droplets indicating the presence of material within the microdroplets.

[0071] In one embodiment of the apparatus provided herein, the stored dataset of a memory module may be configured to store and hold one or more thresholds related to one or more characteristics measured by a detector, and the controller may be configured to select one or more microdroplets having measurement datasets equal to, above, or below one or more thresholds. The thresholds may be set by the user.

[0072] The controller may be configured to select one or more microdroplets and move the selected one or more microdroplets to one or more waste electrowetting paths. For example, the controller may select one or more undesirable microdroplets based on the fact that their measured values ​​are equal to, above, or below one or more stored thresholds set by the user, and move them to one or more waste electrowetting paths.

[0073] In one embodiment, one or more desirable microdroplets having measurements equal to, above, or below one or more stored thresholds set by the user are not selected by the controller and remain within one or more electrowetting paths.

[0074] In one embodiment, if the controller determines that the ether electrode is not occupied by droplets, the ether electrode may be disabled to create additional space for path-to-path redistribution. This can improve the efficiency of redistribution processes such as waste removal or droplet redistribution before the array.

[0075] In one embodiment, an apparatus for manipulating one or more microdroplets into an array using an EWOD or oEWOD may comprise: a chip for manipulating microdroplets having a plurality of electrowetting paths leading to an array and one or more waste electrowetting paths leading to a waste outlet; a detector configured to acquire a measurement dataset related to the distinct characteristics of a detected microdroplet in order to detect one or more microdroplets having distinct characteristics; a storage module configured to store and hold a storage dataset including one or more thresholds related to the characteristics measured by the detector; and a controller configured to receive the storage dataset and acquired measurement datasets from the storage module and determine whether the measurement dataset is equal to, above, or below the thresholds of the storage dataset, wherein the controller is configured to select one or more microdroplets having a measurement dataset that is equal to, above, or below the thresholds of the storage dataset and to move them to the waste electrowetting path.

[0076] In some embodiments of the apparatus described herein, the detector and the memory module may be configured to allow adjustment of the threshold during operation.

[0077] The controller may be configured to select one or more undesirable microdroplets and move them from the first region or electrowetting path to a waste electrowetting path before they reach a second region. Furthermore, the second region may be defined as a collection of array locations such that the second region is in the same range as the array. In this case, the controller may be configured to select one or more undesirable microdroplets and move them to a waste electrowetting path adjacent to the second region. The detour to a waste electrowetting path adjacent to the array ensures that the down-selected microdroplets are detoured at the point where they would join the array. In one embodiment, the second region may include a plurality of subarrays separated by a waste electrowetting path. Thus, the controller may be configured to direct microdroplets to one of the subarrays or to a waste electrowetting path once they enter the second region.

[0078] In some embodiments of the apparatus described herein, the controller may be configured to select one or more undesirable microdroplets and move one or more selected microdroplets from an electrowetting path to a waste electrowetting path. In some embodiments of the apparatus described herein, the controller may be configured to select multiple undesirable microdroplets and move multiple selected microdroplets from multiple electrowetting paths to one or more waste electrowetting paths.

[0079] In one embodiment of the apparatus described herein, the controller may be configured to select one or more undesirable microdroplets and move them from the first region or electrowetting path to a waste electrowetting path before they reach the second region.

[0080] In one embodiment of the apparatus described herein, the controller may be configured to select one or more undesirable microdroplets and move one or more selected microdroplets from a first or second area to a waste electrowetting path.

[0081] In one embodiment of the apparatus described herein, the controller may be configured to select one or more undesirable microdroplets, to move one or more selected undesirable microdroplets into the space between electrowetting paths, to move one or more selected undesirable microdroplets in a direction intersecting one or more electrowetting paths, and to move one or more selected undesirable microdroplets to a waste outlet via a waste electrowetting path.

[0082] By selecting one or more undesirable microdroplets and configuring the controller to move the selected microdroplets in a direction that intersects the electrowetting path, the undesirable microdroplets can be moved without disrupting the flow of microdroplets within the electrowetting path.

[0083] In one embodiment of the apparatus described herein, the detector may be a bright-field imaging detector configured to detect microdroplets and acquire a measurement dataset.

[0084] In one embodiment of the apparatus described herein, the distinct characteristics measured by the detector may be the diameter of the microdroplet, fluorescence, or the transmittance of light passing through the microdroplet.

[0085] In one embodiment, the controller may be further configured to select one or more microdroplets based on an optical label, such as a fluorescent label attached to the microdroplet. In another embodiment, the controller may be configured to select one or more microdroplets containing a fluorescent object or molecule, such as a stained cell or dye.

[0086] In some embodiments of the apparatus described herein, the controller may be configured to select microdroplets having an undesirable size. The distinct characteristic measured by the detector may be the diameter of the microdroplet, and the controller may be configured to select one or more microdroplets having a measurement dataset equal to, above, or below a threshold for microdroplet diameter. The threshold for microdroplet diameter may be 0.5 to 1.5 times the expected microdroplet diameter, or the threshold may be 0.9 to 1.1 times the microdroplet diameter. The selection of this threshold depends on the requirements of the experiment, and in some applications a much smaller range of 0.97 to 1.03 times the diameter may be required, although this may lead to increased loading time.

[0087] In one embodiment of the apparatus described herein, the microdroplets may contain cells, and a distinct characteristic measured by the detector may be the transmittance of light passing through the microdroplet, thereby indicating whether the microdroplet contains the desired cells or is empty. Regions of small intensity changes within the droplet can be detected. By comparing the intensity change with a stored threshold, droplets containing objects can be identified. If the intensity change is greater than the stored threshold, it can be determined that the droplet contains small objects such as cells.

[0088] In one embodiment, the droplet may contain a fluorescent reporter, and the distinct characteristic measured by the detector may be fluorescence, thereby indicating the presence of a fluorescent reporter within the microdroplet or indicating that the microdroplet is empty.

[0089] In some embodiments, detection of one or more desirable microdroplets may be performed using light or optical spectroscopy, such as fluorescence spectroscopy or Raman spectroscopy. In some embodiments, the detector is configured to detect the fluorescence of one or more desirable microdroplets. In some embodiments, the detector may be a fluorescence detector. Additionally or alternatively, the detector may be configured to detect fluorescently labeled undesirable microdroplets.

[0090] In one embodiment of the apparatus described herein, electrowetting paths and / or waste electrowetting paths may be created by a series of moving sprite patterns.

[0091] A sprite pattern is an array of one or more individual sprites, which are highly localized electric fields formed from the photoexcitation of the photoconductive layer of the chip. The number of sprites in a sprite pattern can be any appropriate number and can change over time, as sprites can be added to or removed from the sprite pattern to facilitate the propagation of the sprite pattern and the resulting electrowetting or waste electrowetting paths. In some embodiments of the apparatus described herein, each individual sprite can control a single droplet. This ensures precise control of microdroplets and their organization into an array.

[0092] In some embodiments of the apparatus described herein, one or more electrowetting paths may be configured to split to form two or more electrowetting paths. This may facilitate the handling of droplets and allow for the separation of undesirable droplets from the rest of the droplet array by creating the necessary space between the electrowetting paths for the formation of a waste electrowetting path. In some embodiments, one or more electrowetting paths can be combined together to form at least one additional electrowetting path. Once a waste path is created, the waste path extends parallel to the array path and simultaneously between the array paths. This is important to avoid time-dependent obstacle avoidance calculations and to allow the original loading path to operate at 100% fill rate. Thus, following this methodology allows for highly parallelized loading and selection operations, enabling experiments with a large number of droplets (>100s).

[0093] Furthermore, according to the present invention, an apparatus is provided for manipulating one or more microdroplets to form an array using an EWOD or oEWOD, the apparatus comprising: a) a chip for manipulating microdroplets having a plurality of electrowetting paths leading to an array, and ii) one or more waste electrowetting paths leading to a waste outlet; b) a detector configured to acquire a measurement dataset related to the distinct characteristics of a detected microdroplet in order to detect one or more microdroplets having distinct characteristics; c) a storage module configured to store and hold a storage dataset related to the characteristics measured by the detector; and d) a controller configured to receive the storage dataset and acquired measurement dataset from the storage module and to determine whether the measurement dataset is related to a desirable or undesirable characteristic, wherein the controller is configured to select one or more microdroplets having a measurement dataset related to an undesirable characteristic and to move the one or more selected microdroplets to a waste electrowetting path, and the controller is configured to control the movement of microdroplets along and / or between electrowetting paths so that the movement of microdroplets is synchronized.

[0094] In this context, the term "synchronized" is used to describe the efficient movement of the ether electrodes, i.e., the movement of microdroplets. The movement of microdroplets may be nearly continuous, moving one row at a time across the pixelated grid. For synchronization to occur, the microdroplets do not necessarily have to move in the same direction, and may not move at all. However, when microdroplets move, they move at the same time as other moving microdroplets. Also, the moving microdroplets may move at nearly the same velocity.

[0095] In one embodiment of the apparatus described herein, the controller is configured to form a plurality of electrowetting paths such that the movement of each microdroplet to each of the electrowetting paths can be synchronized with respect to one another. This allows one or more microdroplets to be moved by the controller without disturbing other microdroplets in the electrowetting paths.

[0096] In one embodiment, the method may include moving one or more microdroplets having a measurement dataset associated with an undesirable characteristic, and preventing one or more undesirable microdroplets from forming part of an array.

[0097] In one embodiment, a microfluidic chip according to any aspect of the present invention includes a first composite wall having a first substrate, a first transparent conductive layer on the substrate having a thickness in the range of 70 to 250 nm, a photoactive layer having a thickness in the range of 300 to 1500 nm and activated on the conductive layer by electromagnetic radiation in the wavelength range of 400 to 1000 nm, and a first dielectric layer on the photoactive layer having a thickness in the range of 30 to 160 nm, and a second composite wall having a second substrate, a second transparent conductive layer on the substrate having a thickness in the range of 70 to 250 nm, and optionally a second dielectric layer on the photoactive layer having a thickness in the range of 30 to 160 nm or 120 to 160 nm, wherein the exposure of the first and second dielectric layers The oEWOD structure defines a microfluidic space adapted to accommodate microdroplets, with the exposed surfaces positioned at a distance of less than 180 μm; an A / C power supply connects the first and second conductor layers and supplies a voltage across the entire first composite wall; at least one electromagnetic radiation source having an energy higher than the band gap of the photoactive layer, adapted to collide with the photoactive layer and induce a virtual electrowetting position corresponding to the surface of the first dielectric layer; and means for manipulating the collision point of the electromagnetic radiation onto the photoactive layer so as to change the arrangement of the virtual electrowetting position to create at least one electrowetting path that allows the microdroplets to move.

[0098] In one embodiment, the first dielectric layer and the second dielectric layer may be composed of a single dielectric material, or they may be a composite of two or more dielectric materials. The dielectric layer is made of Al2O3 and SiO2O3. 2 It may be made from, but is not limited to, these materials.

[0099] In some embodiments, a structure may be provided between the first and second dielectric layers. The structure between the first and second dielectric layers may be made of epoxy, polymer, silicon, or glass, or a mixture or composite thereof, and may have straight lines, angles, curves, or microstructured walls / faces.

[0100] The structure between the first and second dielectric layers may be connected to the upper and lower composite walls to create a sealed microfluidic device and define channels and regions within the device. The structure may occupy the gap between the two composite walls.

[0101] In one embodiment, the microfluidic device may be an oEWOD device, which includes a first composite wall having a first substrate, a first transparent conductive layer on the substrate having a thickness in the range of 70 to 250 nm, a photoactive layer having a thickness in the range of 300 to 1500 nm and activated on the conductive layer by electromagnetic radiation in the wavelength range of 400 to 850 nm, and a first dielectric layer on the photoactive layer having a thickness of less than 20 nm, such as between 1 nm and 20 nm; and a second composite wall having a second substrate, a second transparent conductive layer on the substrate having a thickness in the range of 70 to 250 nm, and optionally a second dielectric layer on the photoactive layer having a thickness of less than 20 nm, such as between 1 nm and 20 nm. The oEWOD structure comprises a microfluidic space adapted to accommodate microdroplets, wherein the exposed surfaces of the first and second dielectric layers are positioned at a distance of 20 to 180 μm; an A / C power supply connecting the first and second conductor layers and supplying a voltage across the entire first composite wall; first and second electromagnetic radiation sources having higher energy than the band gap of the photoactive layer, adapted to collide with the photoactive layer and induce virtual electrowetting positions corresponding to the surface of the first dielectric layer; and means for manipulating the collision points of electromagnetic radiation onto the photoactive layer so as to change the arrangement of the virtual electrowetting positions to create at least one electrowetting path that allows microdroplets to move. The first and second walls of these structures are transparent, with the microfluidic space between them.

[0102] Preferably, the first and second substrates are made of a material having mechanical strength, such as glass, silicon, metal, or engineering plastic. In some embodiments, the substrates may have some degree of flexibility. In yet another embodiment, the first and second substrates have a thickness in the range of 100 to 1500 μm, for example, 500 μm or 1100 μm. In some embodiments, the first substrate is composed of one of silicon, fused silica, and glass. In some embodiments, the second substrate is composed of one of fused silica and glass.

[0103] The first and second conductive layers are located on one surface of the first and second substrates and typically have a thickness in the range of 70 to 250 nm, preferably 70 to 150 nm. At least one of these layers consists of a transparent conductive material such as indium tin oxide (ITO), a very thin film of a conductive metal such as silver, or a conductive polymer such as PEDOT. These layers may be formed as a continuous sheet or as a series of discrete structures such as wires. Alternatively, the conductive layers may be a mesh of conductive material, and electromagnetic waves are directed through the gaps in the mesh.

[0104] The photoactive layer is preferably composed of a semiconductor material capable of generating localized regions of charge in response to stimulation by a second source of electromagnetic radiation. An example is a hydrogenated amorphous silicon layer having a thickness in the range of 300 to 1500 nm. In some embodiments, the photoactive layer is activated by the use of visible light. The photoactive layer in the case of the first wall, and optionally the conductive layer in the case of the second wall, are covered with a dielectric layer, typically having a thickness in the range of 1 to 160 nm. The dielectric layer may consist of a single dielectric or be constructed from multiple layers of different dielectrics. The dielectric properties of this layer preferably include a high dielectric strength of >10^7 V / m and a dielectric constant of >3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a thin non-conductive polymer film.

[0105] In another embodiment of these structures, at least the first dielectric layer, preferably both, is coated with an antifouling layer to assist in establishing desirable microdroplet / carrier liquid / surface contact angles at various virtual electrowetting electrode positions, and further to prevent the contents of the microdroplets from adhering to the surface and decreasing as they move through the chip. If the second wall does not constitute the second dielectric layer, the second antifouling layer can be applied directly onto the second conductive layer.

[0106] For optimal performance, the antifouling layer should help establish a microdroplet / carrier liquid / surface contact angle that should be in the range of 50-180 when measured as an air-liquid-surface three-point interface at 25°C. In some embodiments, these layers(s) have a thickness of less than 10 nm and are typically monolayers. In other embodiments, these layers consist of polymers of acrylic acid esters such as methyl methacrylate or their derivatives substituted with hydrophilic groups, such as alkoxysilyls. One or both of the antifouling layers are hydrophobic to ensure optimal performance. In some embodiments, a silica interstitial layer less than 20 nm thick may be interposed between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0107] The first and second dielectric layers, and therefore the first and second walls, have a width of at least 10 μm, preferably in the range of 20 to 180 μm, and define a microfluidic space within which microdroplets are contained. Preferably, the microdroplets themselves, before being contained, have an intrinsic diameter that is 10% or more, preferably 20% or more, larger than the width of the microdroplet space. Thus, upon entering the chip, the microdroplets are subjected to compression, improving electrowetting performance, for example, through better microdroplet confluence ability. In some embodiments, the first and second dielectric layers can be coated with a hydrophobic coating such as fluorosilane.

[0108] In another embodiment, the microfluidic space includes one or more spacers to hold the first and second walls separated by a predetermined distance. Spacer options include beads or pillars, or raised sections created from an intermediate resist layer manufactured by photopatterning. Spacers can also be created using deposited materials such as silicon oxide or silicon nitride. Alternatively, a layer of film containing a flexible plastic film, with or without an adhesive coating, can be used to form the spacer layer. Various spacer shapes can be used to form narrow channels, tapered channels, or partially enclosed channels defined by lines of pillars. With careful design, these spacers can be used to assist in the deformation of microdroplets, followed by the fragmentation of the microdroplets and manipulation of the deformed microdroplets. Similarly, these spacers can be used to physically separate areas of the chip to prevent cross-contamination between droplet clusters and to facilitate the flow of droplets in the correct direction when loading the chip with hydraulic pressure.

[0109] The first and second walls are biased using an A / C power supply attached to the conductive layer, providing a potential difference between them, preferably in the range of 1 to 50 volts. These OEWOD structures are typically employed in conjunction with a second source of electromagnetic radiation having wavelengths in the range of 400 to 850 nm, e.g., 550, 620, and 660 nm, and energies exceeding the band gap of the photoactive layer. Preferably, the photoactive layer is configured such that the incident intensity of the employed radiation is 0.01 to 0.2 Wcm 2 It will be activated at the location of the virtual electrowetting electrode within the range.

[0110] When the electromagnetic radiation source is pixelated, it is preferable to supply it directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated with light from an LED or other lamp. This allows for the rapid creation and destruction of highly complex patterns of virtual electrowetting electrode positions on the first dielectric layer, thereby enabling precise navigation of microdroplets along essentially arbitrary virtual paths using precisely controlled electrowetting forces. Such electrowetting paths can be considered to be constructed from a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0111] The first and second dielectric layers may consist of a single dielectric material, or they may be a composite of two or more dielectric materials. The dielectric layers can be made from Al2O3 and SiO2, but are not limited to these.

[0112] A structure can be provided between the first dielectric layer and the second dielectric layer. The structure between the first and second dielectric layers can be made of epoxy, polymer, silicon, or glass, or a mixture or composite thereof, and may have straight lines, angles, curves, or microstructured walls / faces. The structure between the first and second dielectric layers can be connected to upper and lower composite walls to create a sealed microfluidic device, defining channels and regions within the device. The structure can occupy the gap between the two composite walls. Alternatively, or additionally, the conductor and dielectric can be deposited on a substrate that already has a walled shape.

[0113] In some embodiments of the devices provided herein, one or more microdroplets contain a biological or chemical material different from the microdroplet medium. In some embodiments of the devices provided herein, the microdroplet medium may be a cell medium and may be selected from the following: F12 growth medium, RPMI medium, DMEM, and Opti-MEM or EMEM.

[0114] In some embodiments of the devices provided herein, the biological or chemical material is selected from biological cells, cell culture media, chemical compounds or compositions, drugs, enzymes, beads having materials optionally bound to their surface, or microspheres. In some embodiments, polystyrene or magnetic beads may be bound to antigens, antibodies, or small molecules via biotin-streptodavidin bonds. In some embodiments, oligonucleotides may be bound as DNA tags. In some embodiments, small molecules or dye molecules may be bound with or without the use of a UV-cleavable linker.

[0115] In some embodiments of the devices provided herein, the biological cells may be mammals, bacteria, fungi, yeast, macrophages, or hybridomas, and may be selected from, but are not limited to, CHO, Jurkat, CAMA, HeLa, B cells, T cells, MCF-7, MDAMB-231, Escherichia coli, and Salmonella. In some embodiments of the devices provided herein, the chemical compounds or compositions may include enzymes, assay reagents, antibodies, antigens, drugs, antibiotics, lysis reagents, surfactants, dyes, or cell stains. In some embodiments of the devices provided herein, the biological or chemical materials may be DNA oligos, nucleotides, loaded or unloaded beads / microspheres, fluorescent reporters, nanoparticles, nanowires, or magnetic particles.

[0116] In some embodiments, detection of one or more microdroplets can be performed using optical or light-based spectroscopy, such as fluorescence spectroscopy. In some embodiments, the detector may be configured to detect the fluorescence of one or more microdroplets. In some embodiments, the detector may be a fluorescence detector.

[0117] In a further embodiment of the present invention, a method is provided for manipulating microdroplets to form an array using an EWOD or oEWOD, the method comprising the steps of preparing a device according to any aspect of the present invention, and moving one or more microdroplets toward an array through a plurality of electrowetting paths, wherein the spacing between the electrowetting paths is at least twice the average microdroplet diameter, and the microdroplets move continuously within the electrowetting paths by application of an EWOD or oEWOD force without moving between the electrowetting paths.

[0118] The spacing of the electrowetting pathways must be at least twice the average microdroplet diameter so that a single microdroplet can pass between two other microdroplets. This is necessary to achieve a sieving effect and to allow droplets not controlled by EWOD or oEWOD forces to fall into the gaps between microdroplets controlled by EWOD or oEWOD forces. Thus, this is necessary for self-organized loading. After the droplets have organized into an array, it is also possible to narrow the spacing of the electrowetting pathways to bring the droplets closer together. [Brief explanation of the drawing]

[0119] The present invention will now be described in more detail, with reference to the attached drawings, which are merely illustrative examples. [Figure 1] This diagram shows the flow direction from the inlet to the outlet, with a droplet loaded into the tip described herein. [Figure 2] This is a plot showing the flow velocity within the chip. [Figure 3] Plots A through D show the effect of channel length on flow velocity within the chip. [Figure 4] Plots A through C show the effect of channel-outlet separation distance on flow velocity within the chip. [Figure 5] This plot shows the effect of both channel length and inlet-outlet distance on flow velocity within the chip. [Figure 6] This diagram illustrates how a chip equipped with droplets experiences a sharp decrease in flow velocity at the distal end of the channel, which consequently affects the droplets. [Figure 7] This diagram illustrates a loading scheme in which droplets are loaded onto a chip, and an electrowetting pattern is generated by the application of a temporary EWOD or oEWOD force. [Figure 8] The electrowetting pattern shows the tip passing over the droplet at the distal end of the channel. [Figure 9] This is a chip where droplets have been picked up and aligned by an electrowetting pattern. [Figure 10] This shows a chip where droplets are arranged in an array by an electrowetting pattern. [Figure 11] This shows a series of moving sprite patterns that pick up microdroplets and form electrowetting paths leading to a self-organized array of microdroplets. [Figure 12] This shows an electrowetting path formed by one or more sequences of moving sprite patterns, where the electrowetting path propagates from a first region of the chip at substantially the same angle. [Figure 13] Figure 13A shows a sprite pattern in which sprites are generated at the corners of the sprite pattern as the sprite pattern moves. Figure 13B illustrates how generating sprites at the corners of the sprite pattern causes the sprite pattern to propagate at different angles. Figure 13C shows electrowetting paths propagating in different directions from a first region of the chip. [Figure 14A] This shows how the microdroplets within the electrowetting pathway and the electrowetting pathway itself divide into three separate electrowetting pathways, with space created between them for a waste electrowetting pathway. [Figure 14B] This demonstrates the presence of undesirable droplets alongside desirable ones in the electrowetting pathway. [Figure 14C] This shows how undesirable droplets move to the waste electrowetting path. [Figure 14D] This shows how undesirable droplets are moved away from the waste electrowetting path and traverse the electrowetting path without interfering with desirable droplets within that path. [Figure 14E] This shows how undesirable droplets are moved from the electrowetting pathway to the waste electrowetting pathway. [Figure 14F] This shows how undesirable droplets are moved from the waste electrowetting path to the electrowetting path without interfering with desirable droplets within that electrowetting path. [Figure 14G] This demonstrates that undesirable droplets can be moved away from the electrowetting path and subsequently transferred to the waste outlet via the waste electrowetting path. [Figure 15] This diagram shows how multiple electrowetting paths are branched and divided, and how a waste electrowetting path is created between the divided electrowetting paths to form an electrowetting path. [Figure 16] This illustrates how branching of electrowetting pathways creates enough space between them to form waste electrowetting pathways, thus maintaining the number of initial electrowetting pathways and demonstrating the formation of alternative electrowetting pathways. [Figure 17] The electrowetting path and the waste electrowetting path move side by side from the first region of the chip to the second region of the chip, and the waste electrowetting path moves towards the exit of the chip. [Figure 18] This demonstrates the formation of alternative electrowetting paths, where sprites are created at the corners of the sprite pattern, resulting in electrowetting paths propagating from the first region of the chip at different angles. [Figure 19]a shows the loading of a droplet onto the chip. b shows the transition of the droplet to the oEWOD control via the propagating light pattern of the sprite and the beginning of the droplet's self-alignment. c is a diagram showing the spacing of the electrowetting paths to ensure space for the waste electrowetting paths. d shows an undesirable, oversized droplet that has reached a branching point to move to the waste electrowetting path. e shows an undesirable, oversized droplet that has moved to the waste electrowetting path. f shows the undesirable, oversized droplet continuing to move along the waste electrowetting path, including a change of direction in its path. Detailed description of the drawing

[0120] Figure 1 depicts a tip 10 according to the present invention. The tip 10 comprises a droplet reservoir 12 connected to an inlet end 4 within the tip 10. The reservoir 12 is provided for storing a plurality of microdroplets 200. Microdroplets may contain one or more biological or chemical materials different from the microdroplet medium. Microdroplet media may be cell media containing F12 growth medium, RPMI medium, DMEM, and Opti-MEM or EMEM. Chemical or biological substances contained in the microdroplet medium may be biological cells, cell media, chemical compounds or compositions, drugs, enzymes, beads with substances optionally bound to their surface, or microspheres. More specifically, cells may be mammals, bacteria, fungi, yeast, macrophages, hybridomas, and may be selected from, but are not limited to, CHO, Jurkat, CAMA, HeLa, B cells, T cells, MCF-7, MDAMB-231, Escherichia coli, or Salmonella. The chemical substances contained within the microdroplets may be enzymes, assay reagents, antibodies, antigens, drugs, antibiotics, dissolving reagents, surfactants, dyes, or cell stains. Other biological or chemical materials that may be contained within the microdroplets include DNA oligos, nucleotides, loaded or unloaded beads / microspheres, fluorescent reporters, nanoparticles, nanowires, or magnetic particles.

[0121] Connected to the inlet is a channel 6 designed to load one or more droplets 200 into the tip 10. The distal end 7 of the channel has a first region 8 where droplets can be manipulated via EWOD or oEWOD forces. Furthermore, there is also a second region 202 in the device 10 containing droplets 200 that can be organized as an array. The channel 6 may be blunted or grooved at the distal end 7. Alternatively, the channel 6 may be tapered or have substantially the same width along its entire length. The tip also includes at least one outlet end 2, which allows the flow to be directed from the inlet 4 and the distal end 7 of the channel to the outlet 2, as indicated by the arrows in Figure 1. The tip 10 may consist of two or more outlets 2, and in some embodiments, at least one outlet is located on either side of the channel 6.

[0122] The inclusion of an inlet 4 and an outlet 2 may be important for creating a directional flow on the tip 10, the velocity of which is shown by the plot in Figure 2. As shown in Figure 2, the elongated channel 6 extends substantially in a first direction to a first region 8 of the tip 10. A fluid flow containing one or more microdroplets can be pumped or drawn into the channel 6 from the reservoir 12 at the inlet end 4 of the channel 6. The velocity of the fluid flow at the proximal end 5 of the channel 6 can be relatively fast and constant. As the fluid flow containing one or more microdroplets moves further along the channel 6 toward the distal end 7 of the channel 6, the velocity of the fluid flow containing one or more microdroplets at the distal end 7 of the channel 6 will be substantially lower than the velocity of the fluid flow at the proximal end 5 of the channel 6. In some embodiments, the velocity of the microdroplets at the distal end 7 of the channel 6 may be zero or near zero so that the droplets loaded onto the tip 10 effectively stop or nearly stop at the distal end 7 of the channel 6. The distal end 7 of channel 6 may be blunted or grooved to maximize the reduction in fluid flow rate and / or velocity.

[0123] As shown in the plot in Figure 3, the length of channel 6 is a critical parameter of the tip 10. Figure 3 shows the effect of different channel lengths on the velocity within the tip 10. In Figure 3, outlet 2 is fixed at a position 2.25 mm from inlet 4. The channel length 6 can be recessed 0.2 mm relative to outlet 2, as shown in Figure 3A; extend 0.4 mm into the tip 10 relative to outlet 2, as shown in Figure 3B; extend 1.2 mm into the tip 10 relative to outlet 2, as shown in Figure 3C; and extend 2.2 mm into the tip 10 relative to outlet 2, as shown in Figure 3D. Figure 3C shows that channel 6 needs to extend at least 1.2 mm into the tip 10 relative to the outlet to prevent continuous flow between the distal end 7 of the channel and outlet 2.

[0124] Another important parameter of tip 10 is the distance between the inlet 4 and the outlet 2. Referring to Figures 4A to C, plots are provided showing the effect of the separation distance between the inlet 4 and the outlet 2 on the velocity within tip 10. In Figures 4A to 4C, the length of the channel 6 is fixed. The inlet 4 and the outlet 2 can be separated by a distance of 2.25 mm as shown in Figure 4A, by 1.5 mm as shown in Figure 4B, or by a distance of 0.75 mm as shown in Figure 4C. Figure 4A shows that a minimum separation distance of 2.25 mm between the inlet 4 and the outlet 2 is required for continuous flow between the distal end 7 of the channel and the outlet 2 to effectively prevent the droplet loaded into tip 10 from stopping at the distal end 7 of the channel.

[0125] Figure 5 shows the effect of the combination of channel length 6 and the distance between inlet 4 and outlet 2 on the velocity. When the distance between inlet 4 and outlet 2 is 2.25 mm, a minimum channel length 6 of 1.2 mm is required with respect to outlet 2 to prevent continuous flow between the distal end 7 of the channel and outlet 2. When the distance between inlet 4 and outlet 2 is reduced to 1.5 mm, the required minimum channel length 6 increases to extend 2.2 mm into the tip 10 with respect to outlet 2, while a distance of 0.75 mm between inlet 4 and outlet 2 is unsuitable for use with the channel length 6 examined.

[0126] Once a droplet is loaded into the channel 6 through the inlet 4, the droplet will effectively stop at the distal end 7 of the channel due to a low flow velocity region. The effect of flow velocity on a droplet loaded into tip 10 is shown in Figure 6, which shows that the near-zero flow velocity region causes the droplet to spread out in a fan shape from the distal end 7 of the channel. This near-zero velocity region allows the droplet to stop moving and be removed from flow control, facilitating the transfer of the droplet to EWOD or oEWOD control.

[0127] An example of a method for effectively manipulating and / or controlling droplets from the distal end 7 of the elongated channel 6 within the tip 10 is to create an aligned array using multiple electrowetting paths created by the application of transient EWOD or oEWOD forces at positions along the path, as shown in Figures 7 to 10. To control the droplets using EWOD or oEWOD, a series of EWOD or oEWOD electrowetting patterns 14 are generated, as shown in Figure 7. The electrowetting patterns 14 are shifted over the disordered droplets at the distal end 7 of the channel, as shown in Figure 8, and as the electrowetting patterns 14 pass over the droplets, the droplets are pulled from the distal end 7 of the channel and picked up by the electrowetting patterns 14. The electrowetting patterns 14 move so that the disordered droplets self-assemble as an aligned array, as shown in Figures 9 and 10. Droplets that are not yet controlled by the EWOD or oEWOD forces fall into the gaps of the electrowetting patterns 14, achieving a sieving effect. To obtain this sieving effect, the spacing between the series of electrowetting patterns 14 is at least twice the average microdroplet diameter. After the droplets self-organize as an array, it is possible to narrow the spacing between the electrowetting patterns 14 to move the droplets closer together. The loading and manipulation of droplets using the EWOD or oEWOD described herein can be performed continuously and in parallel.

[0128] Figure 11 illustrates one or more sprite patterns 204 in a first region 8 of the device. One or more sprite patterns 204 can be generated using EWOD or oEWOD forces and can be overlaid at the location of a microdroplet 200 at the distal end 7 of the channel 6, as illustrated in Figure 2. The sprite pattern 204 is shifted over the droplet 200, and the droplet 200 is picked up by the sprite pattern 204 without active detection, achieving efficient passive loading of the droplet 200 onto the sprite pattern 204. Each individual sprite can control one droplet. Individual sprites that do not pick up a microdroplet can be removed. The sprite pattern 204 moves the droplet 200 and, as shown in Figure 11, forms an electrowetting path 206, in which a highly localized electrowetting field can move the microdroplet 200 on the surface of the dielectric layer of the chip 10 by an inductive capillary force.

[0129] While in the electrowetting path 206, droplets 200 that are not yet controlled by the EWOD or oEWOD force fall into the gaps of the sprite pattern 204, achieving a sieving effect. The electrowetting path 206 transports the droplets 200 to a second region of the device 202, where the droplets 200 are organized into an array 208. The loading and manipulation of droplets 200 using the EWOD or oEWOD as described herein can be performed continuously and in parallel.

[0130] For high-speed processing applications, it is necessary to efficiently mount and manipulate millions of microdroplets 200 on a chip 10. It is necessary to be able to manipulate (including controlling the movement, merging, splitting, or changing the shape of microdroplets), sort, and divert the droplets 200 within the chip 10. For example, it is necessary to be able to reuse individual droplets that are deemed undesirable and move these undesirable droplets to outlet 2 within the chip 10 to prevent them from forming part of the droplet array 208.

[0131] In a device designed to process millions of microdroplets 200 at once, the movement and sorting of the microdroplets 200 must make efficient use of space on the chip 10. As shown in Figure 12, one configuration of electrowetting paths 206 that can be used to move and sort the microdroplets 200 while making efficient use of space on the chip 10 includes multiple electrowetting paths 206 propagating at approximately the same angle from a first region 8 of the device. The initial number of electrowetting paths 206 can be the same as the final number of electrowetting paths 206, or the electrowetting paths 206 can branch into two or more electrowetting paths 206, thereby enabling continuous propagation of the electrowetting paths 206 and continuous picking up of droplets 200 from the distal end 7 of the channel 6. The electrowetting paths 206 are created by a controller, which may be a software controller, and each microdroplet 200 within the electrowetting path 206 All other microdroplets The movement of each microdroplet 200 can be configured to be synchronized. This ensures that each microdroplet 200 moves within the electrowetting path 206 without interfering with other microdroplets 200 within the electrowetting path 206.

[0132] The electrowetting path 206 can be activated via a controller to optimize the space used on the chip 10. Microdroplets 200 move continuously within the electrowetting path 206 without moving between them unless activated so via the controller, because a minimum spacing of at least twice the microdroplet diameter is maintained between the electrowetting path 206.

[0133] An alternative embodiment of the electrowetting path configuration can be created by adding sprites to the corners of the sprite pattern 204 as the sprites move over the microdroplet 200 at the distal end 7 of the channel 6 in the first region 8 of the device, as shown in Figure 13A. Adding sprites to the corners of the sprite pattern 204 as the droplet 200 is picked up and loaded onto the sprites creates a sprite pattern 204 that propagates at different angles, as shown in Figure 13B.

[0134] Figure 13C shows multiple electrowetting paths 206 propagating in different directions from a first region 8 of the device, created by a series of moving sprite patterns 204 propagating at different angles. The electrowetting paths 206 propagating in different directions can optimize the use of space on the chip 10 and may allow droplets 200 to be loaded onto the sprite patterns 204 simultaneously in multiple directions.

[0135] Referring to Figure 14A, droplets 200 can be removed from the chip via a waste electrowetting path 300 created by the controller between the electrowetting paths 206. To ensure sufficient space between the electrowetting path 206 and the waste electrowetting path 300, the electrowetting path 206 may be divided into two or more electrowetting paths 207, 209, and 211 to create space for the waste electrowetting path 300 created in between.

[0136] The detector may be configured to identify undesirable droplets 302 from a plurality of droplets 200 flowing along the electrowetting path 211, as shown in Figure 14B. Undesirable droplets 302 may include, but are not limited to, droplets 200 having a diameter above or below a threshold, or droplets 200 that are determined not to contain desired contents or their number, such as particles, chemicals, or biological cells, by measuring their transmittance or fluorescence.

[0137] As shown in Figures 14A to 14G, the multiple electrowetting paths 207, 209, and 211 branch off from their initial shape at angles of 0 to 90°, providing sufficient space for the waste electrowetting paths 301, 303, and 305 formed in between.

[0138] To remove undesirable droplets 302 from the electrowetting path 206 so that they do not form part of the final array 208, the controller may be configured to select one or more undesirable microdroplets 302 and move the selected one or more undesirable microdroplets 302 across a first waste electrowetting path 303, as shown in Figure 14C.

[0139] The controller synchronizes the movement of the undesirable droplet 302 with that of the other droplets 200 in the electrowetting path 206, so that the undesirable droplet 302 can move across the first electrowetting path 209 without disturbing the droplets 200 in the flow within the electrowetting path 209, as shown in Figure 14D.

[0140] The controller can move the unwanted droplet 302 in a direction that intersects with an additional waste electrowetting path 305, as shown in Figure 14E, and an additional electrowetting path 207, as shown in Figure 3F, without disturbing the droplet 200 in the flow within the electrowetting path 207. This process may continue until the unwanted droplet 302 is no longer present between the two electrowetting paths 206, as shown in Figure 14G. The unwanted droplet 302 may then be moved to outlet 2 in the chip 10 via the waste electrowetting path 300.

[0141] Figure 15 is an explanatory diagram of multiple electrowetting paths 206 and multiple waste electrowetting paths 300. The multiple electrowetting paths branch off at one end from their initial formation at angles of 0 to 90° to provide sufficient space for the waste electrowetting paths 300 to form between them, as shown in Figure 15. The spacing between each electrowetting path 206 can be at least twice the average droplet diameter to help reduce or minimize the risk of droplets 200 from different electrowetting paths 206 coming into contact with each other. In one embodiment, the spacing between the electrowetting paths 206 may be at least 100 μm. The electrowetting paths 206 are spread vertically and have a horizontal offset of one array spacing. To remove unwanted droplets 302 from the electrowetting paths 206 at the center of the formation shown in Figure 15, the controller can move across half of the total number of electrowetting paths 206 to remove the unwanted droplets 302 to the waste outlet 300.

[0142] In this embodiment, to prevent unwanted droplets 302 from being carried together with desired droplets 200, the controller can select and remove the unwanted droplets 302 from the chip 10 early in the droplet manipulation process, thereby saving space on the chip 10. The unwanted droplets 302 are removed from the electrowetting path 206 in the first region 8 of the device before reaching the second region of the device 202, and are therefore prevented from forming part of the array 208 in the second region of the device 202.

[0143] In an alternative embodiment, as shown in Figure 16, waste electrowetting paths 300 can be introduced between the electrowetting paths 206 while maintaining the initial number of electrowetting paths 206. The electrowetting paths 206 diverge obliquely from their initial formation at an angle of 0 to 90° until sufficient space is created for the controller to create waste electrowetting paths 300 between the electrowetting paths 206. Electrowetting paths 206 are then formed that extend vertically with a horizontal offset equal to one array spacing.

[0144] One or more undesirable droplets 302 may be moved by the controller to one or more waste electrowetting paths 300 and transported to a second area of ​​the device 202 in the waste electrowetting paths 300. The distance between the electrowetting path 206 and the waste electrowetting path 300 is at least twice the average microdroplet diameter to prevent microdroplets 200 from crossing the paths without action by the controller.

[0145] As shown in Figure 17, the electrowetting path 206 and the waste electrowetting path 300 are aligned parallel to each other from the first region 8 of the device to the second region of the device 202. The electrowetting path 206 carries droplets 200 to form an array 208, while the waste electrowetting path 300 carries unwanted droplets 302 to an exit 2 in the chip 10. Since the waste electrowetting path 300 and the electrowetting path 206 are parallel to each other in the first region 8 and the second region 202 of the chip 10, the controller can select one or more unwanted microdroplets 302 in both the first region 8 and the second region 202 of the chip 10 and move them to the waste electrowetting path 300. Individual sprites of microdroplets 304 that are not being controlled can be removed.

[0146] Both embodiments of the electrowetting paths shown in Figures 15 and 16 show electrowetting paths 206 propagating at substantially the same angle from the first region 8 of the chip 10. According to an alternative embodiment of the device, such as that shown in Figure 18, the electrowetting paths 206 can propagate at different angles from the first region 8 of the chip 10. Sprites are added at the corners of the sprite pattern 204 as the microdroplets 200 are picked up in the first region 8 of the device, causing the sprite pattern 204 to propagate at different angles. The resulting electrowetting paths 206 propagate in different directions, which can optimize the use of space within the chip 10 and allow for simultaneous loading of droplets 200 in multiple directions.

[0147] Filtering of undesirable microdroplets 302 in electrowetting paths 206 propagating at different angles may occur by the same steps illustrated by Figures 14, 15, and 16. Electrowetting paths 206 propagating at different angles from a first region 8 of the device may branch at angles between 0 and 90°, dividing each electrowetting path 206 into two or more electrowetting paths 206, creating sufficient space between them to form a waste electrowetting path 300. Undesirable droplets 302 can be removed from the electrowetting paths 206 propagating at different angles by their movement across the electrowetting paths 206 and waste electrowetting paths 300 in the first region 8 of the device. Alternatively, the electrowetting paths 206 propagating at different angles may diverge at angles between 0 and 90° without dividing the electrowetting paths 206, until sufficient space is created between them to form a waste electrowetting path 300. Unwanted droplets 302 can be removed from the electrowetting path 206 by moving them to a waste electrowetting path 300 in the first region 8 or the second region 202 of the device. The waste electrowetting path 300 can carry the unwanted droplets 302 to an exit 2 in the chip 10.

[0148] Figures 19a to 19f show an example in which an undesirable, oversized microdroplet 302 is selected by the controller and moved to a waste electrowetting path 300. The droplet 200 is loaded onto the chip 10 via channel 6, as shown in Figure 19a, and spreads out in a fan shape from the channel end. The droplet 200 transitions to oEWOD control via the propagating light pattern of sprite 204, and the droplet 200 begins to self-align, as shown in Figure 19b. As shown in Figure 19c, the electrowetting path 206 branches to create space for the waste electrowetting path 300 in between. The undesirable, oversized droplet 302, which is transported within the electrowetting path 206 along with the desired microdroplet 200, reaches the branching point to move onto the waste electrowetting path 300, as shown in Figure 19d. The undesirable microdroplet 302 can be actuated by the controller to move to the waste electrowetting path 300, as shown in Figure 19e. Unwanted oversized droplets 302 can continue to be transported along the waste electrowetting path 300, including when there is a change of direction in its path, and be delivered to the exit 2 in the chip 10, preventing the unwanted droplets 302 from forming part of the array 208.

[0149] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art from the viewpoint of this disclosure.

[0150] As used herein, “and / or” is deemed to be specifically disclosed for each of two designated features or components, whether or not the other is included. For example, “A and / or B” is deemed to be specifically disclosed for each of (i) A, (ii) B, and (iii) A and B, as if each were described separately herein.

[0151] Unless the context indicates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described herein.

[0152] Although the present invention has been described illustratively with reference to several embodiments, it will be further understood by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments can be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

1. A device for manipulating hundreds or thousands of randomly distributed microdroplets into an aligned microdroplet array using EWOD or oEWOD, i) A chip having a first region for receiving and manipulating disordered microdroplets, and a second region for receiving the aligned microdroplet array through a plurality of electrowetting paths providing fluid communication between the first region and the second region, ii) A microdroplet source configured to supply microdroplets, iii) A channel configured to provide fluid communication between the microdroplet source and the first region of the tip, iv) A pressure source configured to move the microdroplets between the microdroplet source and the first region of the tip, Equipped with, The controller is a device configured to enable the synchronous movement of the microdroplets within the electrowetting path by applying an EWOD or oEWOD force.

2. A device according to claim 1, wherein the microdroplet source is a reservoir.

3. A device according to claim 1 or 2, wherein the microdroplet source is a droplet generator.

4. The device according to claim 3, wherein the droplet generator is a step emulsifier.

5. A device according to any one of claims 1 to 4, comprising means for generating an EWOD or oEWOD force configured to generate the electrowetting path by creating one or more moving sprite patterns.

6. The device according to claim 5, wherein each individual sprite controls a single droplet.

7. A device according to any one of claims 1 to 6, wherein the velocity of the microdroplets in the electrowetting path is 25 to 5000 μm / second.

8. A device according to any one of claims 1 to 7, wherein the spacing between the electrowetting paths is 2 to 4 times the average microdroplet diameter.

9. A device according to any one of claims 1 to 8, wherein the distance from center to center of the electrowetting path is at least 100 μm.

10. A device according to any one of claims 1 to 9, wherein the number of existing electrowetting paths is 2 to 250.

11. A device according to claim 10, wherein the number of existing electrowetting paths is 50 to 180.

12. A device according to any one of claims 3 to 11, wherein two or more electrowetting paths propagate from the first region at different angles.

13. A device according to any one of claims 3 to 11, wherein two or more electrowetting paths propagate from the first region at substantially the same angle.

14. A device according to any one of claims 1 to 13, wherein one or more electrowetting paths are divided to form two or more electrowetting paths.

15. A device according to any one of claims 1 to 14, wherein the electrowetting path is created by a controller configured to synchronize the movement of each microdroplet in the path with respect to all other microdroplets.

16. A method for manipulating randomly distributed microdroplets using EWOD or oEWOD to form an aligned microdroplet array, wherein the method is: a) the step of preparing the device described in any one of claims 1 to 15, and b) A step of moving multiple disorganized microdroplets through multiple electrowetting paths to form the aligned microdroplet array, Equipped with, A method wherein the center-to-center distance of the electrowetting path is at least twice the average microdroplet diameter, and the microdroplets move synchronously within the electrowetting path by the application of an EWOD or oEWOD force.

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