Method and apparatus for high throughput droplet manipulation

Abstract

Methods and apparatus are provided for concurrently manipulating and querying the contents of a large number of droplets on a surface of a microfluidic chip. According to one aspect of the invention, there is provided a method for manipulating and inspecting droplets on a microfluidic chip by optically mediated electrowetting (oEWOD), the method comprising: forming a plurality of oEWOD traps on a surface of the chip using a first optical component; forming a second array of oEWOD traps on the surface of the chip using a second optical component; and adjusting the first optical assembly while holding the one or more droplets in place by a second array of oEWOD traps. An apparatus including the microfluidic chip and first and second optical components is also provided.

Description

[0001] This application is a divisional application of the patent application with the national application number 202180012015.8 and the invention title "Methods and Devices for High-Throughput Microdroplet Manipulation", which entered China on July 25, 2022. Technical Field

[0002] The present disclosure relates to methods and devices for manipulating microdroplets, and more particularly to applying optically mediated device electrowetting / dielectrophoretic optical electrowetting (oEWOD) technology to manipulate and interrogate the contents of a large number of microdroplets on the surface of a microfluidic chip in parallel. Background Art

[0003] Dielectrophoretic electrowetting (EWOD) is a well-known effect in which an electric field applied between a liquid and a substrate makes the liquid more wettable on the surface than in its natural state. The effect of electrowetting can be used to manipulate fluids (e.g., move, divide, or change the shape of a fluid) by applying a series of spatially varying electric fields on the substrate to increase surface wettability after a spatially varying sequence. Droplets manipulated in electrowetting-based devices are typically sandwiched between two parallel plates and actuated by digital electrodes. The size of the pixelated electrodes limits the minimum droplet size that can be manipulated and the rate and scale at which droplets can be processed in parallel.

[0004] In our published application WO2018 / 234445 (the entire content of which is incorporated herein by reference), we have described a device for manipulating microdroplets that uses optoelectrowetting to provide motive force. In this optically mediated electrowetting (oEWOD) device, microdroplets are transferred through a microfluidic space defined by containment walls; for example, a pair of parallel plates with a microfluidic space sandwiched therebetween. At least one of these containment walls includes a containment wall hereinafter referred to as a "virtual" electrowetting electrode position, which is generated by selectively irradiating an area of a semiconductor layer buried therein. By selectively irradiating a layer with light from a separate light source controlled by an optical assembly, a virtual path of the virtual electrowetting electrode position can be instantaneously generated, along which the microdroplets can be made to move. Thus, conductive elements are eliminated and permanent droplet receiving positions are dispensed with, which is beneficial for a uniform dielectric surface on which droplet receiving positions are transiently generated by selectively and variably irradiating points on a photoconductive layer using, for example, a pixelated light source. This enables highly localized electrowetting fields that can move microdroplets on the surface by establishing induced capillary-type forces anywhere on the dielectric layer; optionally, in association with any directed microfluidic flow of a carrier medium in which microdroplets are, for example, emulsified and dispersed.

[0005] Another disclosure of oEWOD is the single-sided open configuration platform of "Single-sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns" by Park, Sung-Yong, Michael A. Teitell, and Eric PY Chiou on pages 1655-1661 of Lab on a Chip 10.13 (2010).

[0006] Although these existing platforms allow for fine-grained microdroplet motion control, there are practical limitations (thousands) on the number of microdroplets that can be processed in parallel within a single field of view due to the use of microscope optics to address the sample. However, for some applications, particularly for large-scale screening applications such as those commonly required in the pharmaceutical industry, the need to process droplet numbers on the order of 10^6 or more is required.

[0007] For example, in the fields of cell line development and antibody development, it is necessary to allow for an initial screening of a large number of biological reagents (up to millions) in order to reduce the number of reagents to a sensible number (thousands). To achieve an efficient workflow, this initial screening needs to be performed on a large number of biological agents in a multiplexed manner. Summary of the Invention

[0008] The present disclosure provides methods and related devices in which the flexibility of an oEWOD microfluidic chip is combined with two separately controllable optical components, both of which are capable of generating a fixed but switchable array of light spots on the surface of the oEWOD chip. Such a configuration enables high throughput of microdroplets as well as flexible loading and processing, thus addressing the need for multiplexed processing in screening applications.

[0009] According to the present invention, there is provided a method for inspecting and / or selecting microdroplets on a microfluidic chip by optically mediated electrowetting (oEWOD), the method comprising: temporarily forming a plurality of oEWOD traps on the surface of the chip to cause a plurality of microdroplets on the chip surface to form an array of microdroplets; and maintaining the entire array of microdroplets while inspecting at least one subset of the array.

[0010] Typical implementations of the method of the present invention are cyclic and hierarchical. At a minimum, the method must involve the inspection of droplets or the selection of a subset of droplets. In cases where there is uniformity across a subset of droplets and thus the subset can be automatically selected without inspection, selection without inspection can be appropriate. Inspection without selection is suitable for monitoring the state of multiple droplets. A typical mode of operation would be to inspect the droplets and then select a subset of the inspected droplets based on the information collected from the inspection step. Once selected, the subset of droplets can be held and / or manipulated. The cycle of inspection and selection can then be repeated, and subsequent holding and / or manipulation steps can be performed based on the data collected during inspection.

[0011] Deploying oEWOD traps to create transient arrays offers significant advantages compared to related art that relies on physical structures for containing droplets. The transient structures introduce a significant amount of flexibility to create different structures and modify structures in real time.

[0012] The term oEWOD trap as used herein can also be referred to as a sprite or a fragment. It is light projected onto a surface and it does not contain a fence or a well that is permanently located on the surface. The fragments can form an array, move a portion of the array, and reconstruct a different array at a subsequent moment. Thus, the surface onto which the fragments are projected is effectively a blank canvas, unconstrained by a permanent physical geometry for positioning multiple droplets.

[0013] The ability to hold the entire array of droplets while inspecting a subset of the array allows for the inspection of the array in droplet-level order, down to the droplet, without losing contact with the entire array. By inspecting a subset of the array, optical components with different fields of view can be deployed without losing droplets that are not located within the inspection field of view.

[0014] The step of holding the entire array of droplets can include holding the entire array in a stationary configuration. Alternatively, the holding step can include holding some or all of the array in motion. The traversal can be performed across the surface of the chip while holding some or all of the array. The traversal can be at a substantially constant speed, or it can include deceleration when some or all of the droplets reach a stationary configuration in which they are subsequently held. The step can include capturing droplets that are not attached or locked to the fragments or oEWOD traps.

[0015] The step of holding the entire array of droplets can be facilitated by the following sub-steps: temporarily forming a second array of oEWOD traps on the surface of the chip; and aligning one or more oEWOD traps of the second array with the oEWOD traps of the first array.

[0016] Alignment of the first and second arrays of oEWOD traps enables switching between a first and a second optical component forming the corresponding arrays such that each optical component can inspect or manipulate a subset of the array of microdroplets while the entire array is held in place. In some embodiments, only the second optical component may hold the entire array, while both optical components may inspect and hold subsets of the array.

[0017] Since either of the two components can hold, inspect, and manipulate at least one subset of the array, the responsibility of holding the microdroplets can be switched between the two components in order to optimize the optical performance of the optical component undertaking the detailed inspection and / or manipulation of the subset of microdroplets.

[0018] Typically, one of the optical components has a smaller field of view than the other and is thus capable of holding, inspecting, and manipulating only a subset of the array. The optical component with the smaller field of view will be able to provide more refined manipulation of the droplets within the subset selected due to the improved resolution of the light fragmentation.

[0019] The step of temporarily forming a plurality of OEWOD traps may be performed by the optical component, and the step of temporarily forming a second array of oEWOD traps on the surface may be performed by the second optical component.

[0020] The step of aligning one or more OEWOD traps of the second array with the OEWOD traps of the first array may implement the step of switching between the first and second optical components to hold the entire array of microdroplets.

[0021] In this context, the phrase "entire array" refers to all of the microdroplets currently being held, inspected, and / or manipulated. These droplets may be a uniform linear array occupying the entire surface of the chip. However, when the microdroplets are inspected, merged, separated, and otherwise manipulated, the array may cover only a portion of the chip. Additionally, the droplets may not be a linear array but may be patterned. Furthermore, the phrase "entire array" refers to all of the microdroplets that are operative at that time such that if a subset of microdroplets is deselected and removed, the remaining microdroplets are the entire array at a subsequent time.

[0022] In addition, according to an aspect of the present invention, there is provided a method for manipulating and inspecting microdroplets on a microfluidic chip by optically mediated electro-wetting on dielectric (oEWOD), the method comprising: forming, using a first optical component, a plurality of oEWOD traps on a surface of the chip to cause a plurality of microdroplets on the surface of the chip to form an array of microdroplets corresponding to a first array of oEWOD traps; forming, using a second optical component, a second array of oEWOD traps on the surface of the chip, one or more oEWOD traps of the second array being aligned with the oEWOD traps of the first array; inspecting the contents of the array of microdroplets; and adjusting the first optical component while one or more microdroplets are held in place by the second array of oEWOD traps.

[0023] Having both a first optical component and a second optical component capable of forming an array of OEWOD traps for holding and manipulating microdroplets on the surface of the chip greatly enhances the operational flexibility of the microfluidic chip, as one of these optical components can be used to hold all or a selected portion of the microdroplets in place on the surface while the other component is deactivated or adjusted, meaning that thousands of microdroplets can be manipulated or moved to different positions using different parameters without loss of microdroplets during the interruption required to adjust one of these components.

[0024] In some embodiments, the method further comprises: selecting a subset of microdroplets from the array of microdroplets based on an inspection of the contents of the microdroplets; deactivating all oEWOD traps except those that capture the selected subset of microdroplets; and performing a flushing operation to remove microdroplets that are not in the selected subset from the array of microdroplets.

[0025] The steps of deactivating oEWOD traps for unselected microdroplets (such as those determined to be undesirable during a sorting operation) and performing a flushing operation to remove unwanted microdroplets can be performed on a very large scale with little difficulty, such as in an initial screening assay. For example, the inspection of the contents of the microdroplets can be an inspection to determine which microdroplets are empty and which contain units that are to undergo further observation, and the unselected microdroplets to be flushed can be those that do not contain units. In some embodiments, the flushing operation includes reordering the array of microdroplets using the oEWOD traps of the first optical component such that the removal of microdroplets not in the subset is not hindered by microdroplets in the subset, and / or allowing a continuous phase to enter the microfluidic chip through a plurality of fluid inlets to remove microdroplets not in the selected subset when the associated oEWOD traps have been deactivated.

[0026] The initial steps in the rinsing operation to reorder the array ensure that the removal of unwanted droplets is not hindered by the droplets in the selected subset, and in particular that unwanted microdroplets do not collide with the droplets marked for further inspection during removal. Such reordering can include, for example, switching unwanted microdroplets in the center of the array with those selected for further inspection on the outer edge of the array. Additionally, using a continuous phase to rinse away unselected microdroplets further reduces the need for fine-grained control of microdroplets in large-scale operations, as it is not necessary to use optical components to manipulate unselected droplets across the surface of the chip, but rather to simply maintain the position of the microdroplets in the selected subset by not deactivating the corresponding oEWOD traps. The continuous phase can consist of any one of silicone oil, mineral oil, and fluorocarbon oil.

[0027] In some embodiments, the adjustment of the first optical component includes at least one of the following: a change in resolution, a change in magnification, a change in the field of view, a change in a color selection element included in the component, and replacement of the lens assembly closest to the sample being imaged. In some embodiments, the method further includes: after making the adjustment, using the first optical component to perform further inspection of the contents of the array of microdroplets.

[0028] As described above, the method of the present invention enables greater operational flexibility in microdroplet assays, particularly for large-scale microdroplet operations. In some embodiments, the method of the present invention enables adjustment of assay parameters without loss of any microdroplets from the initially formed array. For example, a first assay can be performed using a first optical component having a wide field of view on an array including thousands of microdroplets, a subset of those microdroplets can be selected for further inspection, and then a second optical component can hold the selected microdroplets in place while reducing the field of view of the first optical component to allow for more precise droplet manipulation or inspection. This example is not limiting, and the principle of holding the selected subset or all of the microdroplets in an array of microdroplets in place while adjusting the parameters of the first optical component between successive assays can be applied to improve efficiency and fine-tune microdroplet assays in various ways.

[0029] In some embodiments, the first optical component according to the present invention as disclosed herein can be used to move, merge, and / or split the microdroplets.

[0030] In some embodiments, the second optical component according to the present invention as disclosed herein can be used to manipulate microdroplets, such as moving, merging, and / or splitting the microdroplets on a microfluidic chip by optically mediated electro-wetting (oEWOD).

[0031] In some embodiments, the method further includes deactivating the first optical component and translating an array of microdroplets across the surface of the microfluidic chip using an OEWOD trap formed by the second optical component. The microfluidic chip typically includes a plurality of different regions that are designed to perform specific operations therein. For example, the surface of the microdroplets may include sorting regions, inspection regions, or regions of the chip surface that have been treated to be suitable for assays using specific types of cells. Another way to improve the operational flexibility of such assays can be achieved by using a dual-component configuration in which the same array of microdroplets or a selected subset thereof is transported between such regions by the second optical component before or after performing the assay. Translating the entire array of oEWOD traps using the second optical component has the advantage that fine-grained droplet control by the oEWOD traps is not required and the relative positions of the droplets in the transported array are maintained relative to each other.

[0032] In some embodiments, the first optical component has a higher imaging resolution than the second optical component. In the dual-component configuration of the present invention, it may be advantageous to have one optical component designated as the creator of the "holding array" that is capable of holding and transporting a large number of microdroplets at once, and another optical component responsible for the high-resolution, adjustable array that is suitable for achieving fine-grained control of the microdroplets in the array when necessary.

[0033] In some embodiments, the step of forming an array of microdroplets includes the following initial steps: forming a plurality of oEWOD traps in the shape of a target array using the second optical component; determining the positions of a plurality of microdroplets on the surface of the microfluidic chip using the first optical component; and manipulating the plurality of microdroplets into an array that matches the target array of oEWOD traps using the plurality of oEWOD traps formed by the first component. Although this operation requires precise droplet control, it is a reliable method for array formation in some chip configurations.

[0034] In other embodiments, the step of forming an array of microdroplets includes: forming a first array of oEWOD traps using the first optical component; and loading a plurality of microdroplets onto the chip surface where the first array of oEWOD traps is located. Forming an array of oEWOD traps on the surface of the chip such that the microdroplets located on the surface of the chip coalesce into the oEWOD trap array positions is an effective method for forming an array of microdroplets in assays involving a large number of microdroplets to be analyzed because this method eliminates the need for precise droplets controlled by droplets during the loading phase. In this way, an array of thousands of microdroplets can be formed quickly and easily.

[0035] In some embodiments, the electromagnetic radiation from the first optical component is multiplexed with the electromagnetic radiation from an inspection component configured to inspect the microdroplets and their contents. Combining the inspection component with the optical components for forming the oEWOD traps allows for more efficient guidance of the inspection radiation.

[0036] In some embodiments, the inspection of the contents of the microdroplets is performed using at least one of the following: fluorescence imaging, local optical plasmon resonance on metal nanoparticles, FRET, dark field, bright field, Raman, absorption, quantum dot fluorescence, spectroscopy. Fluorescence-based methods are particularly advantageous. In the case where the interrogation metric is local optical plasmon resonance on metal nanoparticles, the particles are functionalized with an antigen or an antibody, and the detection method detects the spectral response of the functionalized nanoparticles in response to changes in the binding of the target molecule to the surface.

[0037] In some embodiments, at least one of the first array and the second array is formed using a projection optical device composed of at least one of the following: a spatial light modulator, such as a TFT, a DMD projector, a DLV, and an LCoS projector; a light-emitting array, such as an OLED, a CRT, a projector with a screen, and a micro-LED array.

[0038] According to another aspect of the present invention, there is provided a device for manipulating microdroplets, comprising: a microfluidic chip including a first composite wall and a second composite wall defining a microfluidic space, and the microfluidic chip being configured to manipulate microdroplets on the surface defining the microfluidic space by optically mediated electro-wetting (oEWOD); a first optical component configured to form a first plurality of oEWOD traps to manipulate a plurality of microdroplets on the surface; a second optical component configured to form a second plurality of oEWOD traps on the surface to maintain the relative positions of the plurality of microdroplets during the adjustment of the first optical component and / or during a loading operation; and an inspection component configured to interrogate the contents of the plurality of microdroplets.

[0039] In some embodiments, the inspection component is an electromagnetic radiation source and is multiplexed with the electromagnetic radiation from the first optical component.

[0040] In some embodiments, the first composite wall and the second composite wall are at least partially transparent, and the first optical component and the second optical component are located on opposite sides of the microfluidic space. In other embodiments, at least one of the first composite wall and the second composite wall is transparent, the first optical component and the second optical component are located on the same side of the microfluidic space, and a color filter is applied to the second optical component to prevent interference with the first optical component.

[0041] In some embodiments, at least one of the first optical component and the second optical component includes a microlens array. In some embodiments, the second optical component or both optical components have relatively coarse-grained optical control; rather than switching each illumination spot independently, they are arranged in small groups that can be actuated individually.

[0042] In some embodiments, the device further includes a plurality of sets of external flow control valves and pumps and an arrangement of inlets for loading and flushing operations.

[0043] In some embodiments, the optical component is configured to provide a spot array having spots formed with a pitch between 20 µm and 250 µm and between 50 µm and 675 µm, particularly a spot array having a diameter between 30 µm and 250 µm and formed with a pitch between 30 µm and 300 µm. The pitch is typically 2.5 times the droplet diameter. In some embodiments, the optical component is configured to provide a spot array having an approximate pitch of 100 µm or 125 µm and a spot size of approximately 50 µm. In some embodiments, the optical component is configured to provide a spot array having spots formed with a pitch between 5 µm and 30 µm and between 12.5 µm and 75 µm.

[0044] In some embodiments, the method of the present invention is applied to optically activated devices, such as devices configured to manipulate microparticles (including microparticles in droplets) by dielectrophoresis. Functionally identical optical instruments are used to manipulate and inspect cells or particles to generate a virtual optical dielectrophoresis gradient. Microparticles as defined herein can refer to particles such as biological cells, microbeads made of materials including polystyrene and latex, magnetic microbeads, or colloids.

[0045] Similar to the method for optical electro-wetting described above, a first high-resolution optical component is used to perform fine manipulation and detailed inspection of particles and / or cells through a combination of optically mediated dielectrophoresis. A coarser second optical component is used to form an array of dielectrophoretic traps. The combination of these two components provides the method with the ability to perform fine manipulation and inspection operations using the fine optical component while using the coarser optical component to hold and transport a very large number of particles and / or cells.

[0046] Accordingly, in another aspect of the present invention, there is provided an apparatus for manipulating microparticles, the apparatus comprising: a chip including a first transparent composite wall and a second transparent composite wall, the first and second transparent composite walls defining a holding space and configured to manipulate microparticles located on the surface defining the holding space; a first optical assembly configured to direct an optical beam through the first composite wall onto the surface to form a first plurality of optical traps for manipulating a plurality of microparticles on the surface; a second optical assembly configured to direct an optical beam through the second composite wall onto the surface to form a second plurality of optical traps on the surface for maintaining the relative positions of the plurality of microparticles during adjustment of the first optical assembly and / or during a loading operation; and an inspection component configured to interrogate the contents of the plurality of microparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A cross-sectional view of an example microfluidic chip as described in WO2018 / 234445 is shown;

[0048] Figure 2 A cross-sectional view of an example microfluidic chip suitable for performing the method of the present invention is shown;

[0049] Figure 3 A top view of the surface of the microfluidic space is shown;

[0050] Figures 4A to 4E An example of the process of a loading operation and a droplet interrogation workflow is shown;

[0051] Figure 5A and Figure 5B An illustration of a merging operation of droplets according to the present invention is provided;

[0052] Figure 5C and Figure 5D An illustration of a splitting operation of droplets according to the present invention is provided;

[0053] Figure 6A and Figure 6B An alternative illustration of a merging operation of droplets according to the present invention is provided; and

[0054] Figure 6C and Figure 6D An alternative illustration of a splitting operation of droplets according to the present invention is provided. DETAILED DESCRIPTION

[0055] To further explain various aspects of the present disclosure, specific embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings.

[0056] Reference Figure 1, showing a cross-sectional view of an exemplary microfluidic chip device (as described in WO2018 / 234445) including an oEWOD structure adapted for rapid manipulation of aqueous microdroplets.

[0057] The device includes a top glass plate 13 and a bottom glass plate 14, each 500 µm thick and coated with a 130 nm thick transparent conductive indium tin oxide (ITO) layer 15. Each ITO layer 15 is connected to an A / C source 16, with the ITO layer on the bottom glass plate 14 grounded. The bottom glass plate 14 is coated with an 800 nm thick amorphous silicon layer 17. The top glass plate 13 and the amorphous silicon layer 17 are each coated with a 160 nm thick high-purity alumina or hafnium dioxide layer 18, which is in turn coated with a silica interlayer that supports a trichloro(1H,1H,2H,2H-perfluorooctyl)silane layer 19 to render the surface of the alumina / hafnium dioxide layer 18 hydrophobic. The top glass plate 13 and the amorphous silicon layer 17 are spaced 80 µm apart using spacers, such that microdroplets are subject to a degree of compression when introduced into the device.

[0058] An image of a reflective pixelated screen illuminated by a first optical component (in this example an LED light source) 20 is typically positioned beneath the bottom glass plate 14, and visible light (wavelength 660 nm or 830 nm) at a level of 0.01 W cm−2 is emitted from each diode 21 and irradiates the amorphous silicon layer 17 by propagating in the direction of a plurality of upward arrows through the bottom layer 14 and 15. At each point of incidence, photoexcitation regions 22 that generate charge are created in the amorphous silicon layer 17, which causes modified liquid-solid contact angles in the alumina / hafnium dioxide layer 18 at the corresponding electrowetting positions 23 to create oEWOD traps at these positions. The modified characteristics of the oEWOD trap positions provide the capillary forces required to hold the microdroplets 2 in place or to advance the microdroplets 2 from one point 23 to another. The optical component 20 is controlled by a microprocessor 24, which determines which diodes 21 in the array are illuminated at any given time via a pre-programmed algorithm.

[0059] Reference Figure 2 , showing a cross-sectional view of a microfluidic chip having the same or a similar stacked structure to the Figure 1 chip, where in addition to the first optical component 20, a separately controllable second optical component 25 has been introduced to provide increased flexibility and capabilities for operations involving the handling of large numbers of microdroplets 2.

[0060] The light source from the second optical component can be activated and aligned to the position of the microdroplets held by the first light source during the loading option, and / or can act as a holding light source during the adjustment of the first optical component, e.g., during the switching of the lens in the first light source, during the interrogation, or during the translation of the microdroplet array across the surface of the microfluidic space.

[0061] The light source of the optical component does not need to always be an LED light source. Any optical arrangement that can be used to project an array of programmable light spots in the photoactive layer would be suitable. For example, the projection optics can consist of an LED or LCD screen combined with a microlens array arrangement or a Fly'Eye arrangement. In the examples of the present disclosure, spatial modulation of the illumination pattern for electro-wetting is performed across the object plane (i.e., the plane of the surface on which the oEWOD traps are formed), using, for example, digital micromirror devices, LCD displays, spatial light modulators, or LED arrays. An example projected spot array can consist of dots with a diameter of 50 um with a pitch of 100 um (i.e., the center-to-center distance between the spots).

[0062] In some examples, Figure 2 the device includes a combined inspection and manipulation optical component, where the light suitable for electro-wetting manipulation is multiplexed with the light suitable for fluorescence excitation. Alternatively, the inspection and manipulation components can be physically separated, e.g., if the interrogation component is not an electromagnetic radiation source but a passive collection system.

[0063] Reference Figure 3 , shows a top view of the surface of a microfluidic space that has been loaded with a large number of microdroplets. In this example, the field of view of the first optical component 20 (i.e., the area of influence exerted on the microdroplets 2 on the surface) is shown by the first boundary 26, and the field of view of the second optical component 25 is shown by the second boundary 27. In Figure 3 the example configuration of, it can be seen that the first optical component has a much narrower field of view and is thus more suitable for fine manipulation of a smaller number of microdroplets, while the second optical component has a much wider field of view to be suitable for holding in place a large number of microdroplets traversing most of the surface.

[0064] Generally, the inspection component has an objective lens that has a high numerical aperture suitable for maximizing the light collection efficiency and the resolution of fluorescence imaging. By switching this objective lens, the imaging magnification can be increased or decreased during the determination of the content of the microdroplets in the chip, and concomitantly, the resolution and collection efficiency can be increased or decreased. Increasing the magnification may require reducing the field of view of the imaging system.

[0065] In the case where the inspection optical system and the electro-wetting manipulation optical system are multiplexed, the reduction in the field of view will cause the droplets held in place by the manipulation pattern to be outside the field of view of the optical element; during this period, they can move away by diffusion or fluid flow.

[0066] Similarly, the process of changing the objective lens results in a temporary interruption of the manipulation pattern, during which the droplets may flow away and be lost. Additionally, in some cases, it may be necessary to interrupt the optical manipulation light during fluorescence imaging to prevent light from the manipulation pattern from interfering with the fluorescence image; during such a long interruption, the droplets may move again in an uncontrolled manner.

[0067] These limitations can be overcome by combining a low-resolution spot generation optical component with a high-resolution inspection and manipulation component.

[0068] In an exemplary process, a high-resolution optical component is used to position the droplets into a specific layout in a microfluidic chip, and then the droplets are imaged. Then a pattern aligned with the droplet position is generated on the low-resolution spot generation component. Then, when the objective lens of the inspection component is replaced, this pattern from the low-resolution component can be activated as a holding pattern; the pattern can also be used to hold droplets that are ultimately outside the field of view, and the pattern can be used to hold the droplets during fluorescence acquisition. Alternatively, in some embodiments, the droplets can be positioned into a specific layout by a low-resolution optical component and inspected by a high-resolution optical component.

[0069] The process can be controlled by generating a pixel map on a target surface from two illumination sources through software to create a 2D coordinate transformation between the two sources, which is then applied to the high-resolution source. The coordinate transformation takes into account the pixel scaling and displacement between the two projectors and the different objective lens ranges for the high-resolution source.

[0070] If a broadband light source is selected for the low-resolution optical component, interference with the light used for fluorescence imaging can be eliminated by applying a blocking "notch" filter to the input light, which allows light outside the frequency band to be used for the holding pattern while removing the frequency band of the spectrum.

[0071] Furthermore, the low-resolution optical component can be used as a holding mechanism to maintain the relative positions between the droplets when the sample is moved. For example, if a mechanical motorized motion platform is needed to translate the sample, the pattern registration can be moved such that the droplets move almost in unison with the platform in a stepwise manner, so that their relative positions remain unchanged.

[0072] To overlap two electrowetting control patterns (from the high-resolution component and the low-resolution component), a substantially transparent electrowetting device is preferably used such that a pattern can be projected from each side of the device. Alternatively, the low-resolution pattern can be applied from the same side as the high-resolution pattern but at an oblique angle such that the light enters outside the numerical aperture of the objective lens of the high-resolution component. In this case, a compensating optical element is preferably added to the low-resolution projector to adjust the shape and focal length of the projected pattern, thereby avoiding image distortion caused by projection at an oblique angle.

[0073] Reference Figures 4A to 4E shows another exemplary process of a loading operation and a droplet interrogation workflow, which is suitable for handling a large number of microdroplets, such as more than one million microparticles, without the need for precise droplet-by-droplet control.

[0074] Reference Figure 4A In the first step, the light spot projected in an array shape by the optical component causes photoexcitation of the photoactive layer, resulting in charge accumulation on the surface of the dielectric layer, which serves as an optical trap (oEWOD trap) for the microdroplets on the surface of the microfluidic space within the microfluidic chip.

[0075] See Figure 4B In the second step, the dripping biological agent (such as cells) is introduced into the device under flow in a continuous phase that can consist of an oil (such as silicone oil, mineral oil, or fluorocarbon oil). The interaction between the microdroplets containing the biological agent and the optical trap causes the microdroplets to self-align and merge into a pattern that matches the array pattern of the projection optical device (optical component).

[0076] Reference Figure 4C The array of captured microdroplets is then interrogated through the transparent substrate of the microfluidic chip, and the results of the interrogation are used to select a subset of the microdroplets based on a predetermined metric. The metric can be, for example, fluorescence imaging on metal nanoparticles or surface plasmon resonance detection.

[0077] Reference Figure 4D When a subset of the microdroplets has been selected, the light spots corresponding to the unwanted microdroplets that are not in the selected subset are turned off, and the corresponding microdroplets are removed from the interrogation area on the surface of the chip. Optionally, the corresponding microdroplets are completely removed from the microfluidic chip under a continuous flow that is introduced into the microfluidic chip through a series of pumps and valves for this purpose.

[0078] Reference Figure 4E The set of selected microdroplets then flows out of the chip for further post-processing or analysis, or is retained on the chip for further processing and analysis using the arrangement previously described for controlling the light spots and the movement of the microdroplets using a micro-mirror array.

[0079] In addition to Figure 1 the specific examples provided and illustrated, the general characteristics and optional features of an oEWOD microfluidic chip suitable for performing the method of the present invention, as well as the composite structures included therein, will now be described.

[0080] The oEWOD structure includes: a first composite wall composed of a first substrate and a first transparent conductor layer on the substrate, the first transparent conductor layer having a thickness in the range of 70 nm to 250 nm; a photoactive layer activated by electromagnetic radiation with a wavelength range of 400 nm to 850 nm on the conductor layer, the photoactive layer having a thickness in the range of 300 nm to 1500 nm and a first dielectric layer located on the photoactive layer, the first dielectric layer having a thickness in the range of 30 nm to 160 nm; a second composite wall composed of: a second substrate; a second conductor layer on the second substrate, the second conductor layer having a thickness in the range of 70 to 250 nm, and optionally a second dielectric layer on the second conductor layer, the second dielectric layer having a thickness in the range of 30 nm to 160 nm, wherein the exposed surfaces of the first dielectric layer and the second dielectric layer are arranged to be separated by 20 µm to 180 µm to define a microfluidic space suitable for containing microdroplets; an A / C source for providing a voltage between the first composite wall and the second composite wall connecting the first conductor layer and the second conductor layer; a first electromagnetic radiation source and a second electromagnetic radiation source having higher energy than the bandgap of the photoactive layer suitable for incident on the photoactive layer to cause corresponding virtual electro-wetting positions on the surface of the first dielectric layer; and means for manipulating the incident point of the electromagnetic radiation on the photoactive layer to change the virtual electro-wetting positions so as to create at least one electro-wetting path along which the microdroplets can move. The first wall and the second wall of these structures are transparent, with the microfluidic space sandwiched between the first wall and the second wall.

[0081] Suitably, the first substrate and the second substrate are made of mechanically stronger materials, such as glass, metal or engineering plastics. In some embodiments, the substrate can have a certain degree of flexibility. In yet another embodiment, the first and second substrates have a thickness in the range of 100 µm to 1000 µm. In some embodiments, the first substrate comprises one of silicon, fused quartz and glass. In some embodiments, the second substrate comprises one of fused quartz and glass.

[0082] The first conductor layer and the second conductor layer are located on one surface of the first substrate and the second substrate and generally have a thickness in the range of 70 nm to 250 nm (preferably 70 nm to 150 nm). At least one of these layers is made of a transparent conductive material (such as indium tin oxide (ITO)), a very thin conductive metal film (such as silver) or a conductive polymer (such as PEDOT), etc. These layers can be formed as a continuous sheet or a series of discrete structures, such as lines. Alternatively, the conductor layer can be a grid of conductive material through which electromagnetic radiation is guided between the voids of the grid.

[0083] The photoactive layer suitably comprises a semiconductor material that can generate local charge regions in response to a stimulus by a second electromagnetic radiation source. Examples include a layer of hydrogenated amorphous silicon having a thickness in the range of 300 nm to 1500 nm. In some embodiments, the photoactive layer is activated by using 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 coated with a dielectric layer, typically having a thickness ranging from 30 nm to 160 nm. The dielectric properties of this layer preferably include a high dielectric strength of >10^7 V / m and a dielectric constant of >3. Preferably, it is as thin as possible while avoiding dielectric breakdown. In some embodiments, the dielectric layer is selected from alumina, silica, hafnium oxide, or a thin non-conductive polymer film.

[0084] In another embodiment of these structures, at least the first dielectric layer (preferably both) is coated with an anti-fouling layer to help establish a desired droplet / carrier fluid / surface contact angle at various virtual electro-wetting electrode locations and further to prevent the contents of the droplet from adhering to the surface and being reduced as the droplet moves through the chip. If the second wall does not include a second dielectric layer, a second anti-fouling layer can be applied directly to the second conductor layer.

[0085] To obtain optimal performance, the anti-fouling layer should help establish such a droplet / carrier fluid / surface contact angle that should be in the range of 50 to 180 when measured as an air-liquid-surface three-point interface at 250C. In some embodiments, these layers have a thickness of less than 10 nm and are typically monolayers. In another embodiment, these layers comprise a polymer of acrylate (such as methyl methacrylate or a derivative thereof substituted by a hydrophilic group (e.g., alkoxysilyl)). One or both of the anti-fouling layers are hydrophobic to ensure optimal performance. In some embodiments, a spacer layer of silica having a thickness of less than 20 nm can be placed between the anti-fouling coating and the dielectric layer to provide a chemically compatible bridging portion.

[0086] The first dielectric layer and the second dielectric layer and thus the first wall and the second wall define a microfluidic space having a width of at least 10 μm and preferably in the range of 20 μm to 180 μm, and the droplet is contained in this microfluidic space. Preferably, before the droplet is contained, the droplet itself has an inherent diameter that is more than 10% larger, suitably more than 20% larger, than the width of the droplet space. Thus, upon entering the chip, the droplet is subjected to compression, resulting in enhanced electro-wetting performance through, for example, better droplet coalescence ability. In some embodiments, the first dielectric layer and the second dielectric layer are coated with a hydrophobic coating, such as fluorosilane.

[0087] In another embodiment, the microfluidic space includes one or more spacers for separating the first wall and the second wall by a predetermined amount. Options for the spacers include beads or pillars, ridges created from an intermediate resist layer that has been generated by photolithography. Alternatively, deposited materials such as silicon oxide or silicon nitride can be used to create the spacers. Alternatively, thin film layers (including flexible plastic films with or without an adhesive coating) can be used to form the spacer layer. A variety of spacer geometries can be used to form narrow channels, tapered channels, or partially enclosed channels defined by rows of pillars. Through careful design, these spacers can be used to assist in the deformation of the microdroplets, followed by microdroplet splitting and effect manipulation of the deformed microdroplets. Similarly, when the chip is loaded under hydraulic pressure, these spacers can be used to physically separate the various regions of the chip to prevent cross-contamination between droplet populations and to facilitate the flow of droplets in the correct direction.

[0088] The first wall and the second wall are biased using an AC power source attached to the conductor layer to provide a voltage potential difference therebetween; suitably in the range of 10 to 50 volts. These oEWOD structures are typically used in association with a second electromagnetic radiation source having a wavelength in the range of 400 nm to 850 nm (preferably 660 nm) and an energy that exceeds the bandgap of the photoactive layer. Suitably, the photoactive layer will be activated at the virtual electrowetting electrode locations where the incident intensity of the radiation used is in the range of 0.01 to 0.2 Wcm-2.

[0089] In the case where the electromagnetic radiation source is pixelated, a reflective screen such as a digital micromirror device (DMD) illuminated by light from an LED or other lamp is used to supply the electromagnetic radiation directly or indirectly. This enables highly complex patterns of virtual electrowetting electrode locations to be rapidly created and destroyed on the first dielectric layer, such that the microdroplets can be precisely manipulated along substantially any virtual path using tightly controlled electrowetting forces. Such electrowetting paths can be considered to be constituted by a continuum of virtual electrowetting electrode locations on the first dielectric layer.

[0090] The incident points of the electromagnetic radiation source on the photoactive layer can be of any convenient shape, including conventional circles or rings. In some embodiments, the morphology of these points is determined by the morphology of the corresponding pixels, while in another embodiment, once the microdroplets enter the microfluidic space, the morphology of these points corresponds in whole or in part to the morphology of the microdroplets. In one embodiment, the incident points and thus the electrowetting electrode locations can be crescent-shaped and are oriented in the expected direction of travel of the microdroplets. Suitably, the electrowetting electrode locations themselves are smaller than the surface of the microdroplet adhered to the first wall and give a maximum field strength gradient at the contact line formed between the microdroplet and the surface dielectric.

[0091] In some embodiments of the oEWOD structure, the second wall further includes a photoactive layer that enables the induction of virtual electro-wetting electrode positions on the second dielectric layer by the same or different electromagnetic radiation sources. The addition of the second dielectric layer enables the wetting edge of the microdroplet to transition from the upper surface to the lower surface of the structure and applies more electro-wetting force to each microdroplet.

[0092] The first dielectric layer and the second dielectric layer can be composed of a single dielectric material, or it can be a composite of two or more dielectric materials. The dielectric layer can be made of, but not limited to, Al2O3 and SiO2.

[0093] A structure can be provided between the first dielectric layer and the second dielectric layer. The structure between the first dielectric layer and the second dielectric layer can be made of, but not limited to, epoxy resin, polymer, silicon, or glass or a mixture or composite thereof, having straight, inclined, curved, or microstructured walls / surfaces. The structure between the first dielectric layer and the second dielectric layer can be connected to the top composite wall and the bottom composite wall to produce a sealed microfluidic device and define channels and regions within the device. The structure can occupy the gap between the two composite walls. Alternatively or additionally, conductors and dielectrics can be deposited on a shaped substrate that already has walls.

[0094] Some aspects of the methods and devices of the present invention are suitable for application to optical activation devices different from electro-wetting devices, such as devices configured to manipulate microparticles by dielectrophoresis or optical tweezers. In such devices, optical instruments with the same functions are used to manipulate and inspect cells or particles to generate virtual dielectrophoretic gradients. Microparticles as defined herein can refer to microparticles such as biological cells, microbeads made of materials including polystyrene and latex, hydrogels, magnetic microbeads, or colloids. Dielectrophoresis and optical tweezer mechanisms are well known in the art and can be easily implemented by those skilled in the art.

[0095] Similar to the method for optical electro-wetting described above, a first high-resolution optical component is used to perform fine manipulation and detailed inspection of particles and / or cells through a combination of optically mediated dielectrophoresis. A rough second optical component is used to form an array of dielectrophoretic traps. The combination of these two components provides the ability of the method to perform fine manipulation and inspection operations using the fine optical component while using the rough optical component to hold and transport a very large number of particles and / or cells.

[0096] Reference Figure 5A and Figure 5B , provides an illustration of the merging operation of using a second optical device as described herein to hold droplets within a large area 52 and using a first optical device as disclosed herein to merge the droplets within a smaller field of view 54. Figure 5A Droplets before merging are shown. As Figure 5AThe arrows shown illustrate the direction in which the droplets are merged together. Figure 5B The merged droplets after the merging operation are shown.

[0097] Reference Figure 5C and Figure 5D provide an illustration of a splitting operation in which droplets are held within a large area 52 using a second optical device as described herein and split within a smaller field of view 54 using a first optical device as described herein. As Figure 5C the arrows shown illustrate the direction of splitting in which droplets provide additional droplets. Figure 5D The post-splitting events after the splitting operation are shown.

[0098] Reference Figure 6A and Figure 6B provide an illustration of a merging operation in which droplets are held between operations of a second optical device 52 and merged during the operation of using the second optical device 52, as disclosed herein. Figure 6A The arrows indicated in Figure 6B illustrate the direction of the droplets during the merging operation.

[0099] Reference Figure 6C and Figure 6D provide an illustration of a splitting operation of droplets held between operations of a second optical assembly 52 and splitting the droplets during the splitting operation using the second optical assembly. Figure 6C The arrows indicated in Figure 6D illustrate the direction in which the droplets split to form additional droplets during the splitting operation.

[0100] The optical component for inspecting a subset of the array has a much smaller field of view than the optical component that holds the array in place. Within the reduced field of view, there may be only one microdroplet. Alternatively, there may be 24, 48, 256, 1048, or any suitable number of microdroplets in the field of view of the inspection optical component. The inspection can be performed droplet by droplet, where the optical component scans through its field of view to sequentially inspect each microdroplet. This can involve the optical device being in a single position, and the scanning involves inspecting a portion of the FOV by processing information from a portion of the image that falls on an imaging sensor (such as a camera that forms part of the optical component). Alternatively or additionally, the optical component can be integrated across its entire field of view to obtain an overview of the proportion of microdroplets emitted. This coarse-grained data can be combined with the microdroplets through microdroplet review in order to quickly focus on the most informative part of the array.

[0101] Given the present disclosure, those skilled in the art will appreciate various additional aspects and embodiments of the present invention.

[0102] As used herein, "and / or" shall be taken to mean each specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" shall be taken to mean (i) A, (ii) B, and (iii) each of A and B, as if each were set forth individually herein.

[0103] Unless the context otherwise requires, the descriptions and definitions of the above features are not limited to any specific aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0104] Those skilled in the art will also understand that although the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments, and alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

1. An apparatus for manipulating microdroplets, comprising: A microfluidic chip, the microfluidic chip including a first composite wall and a second composite wall that define a microfluidic space, and the microfluidic chip being configured to manipulate microdroplets on a surface defining the microfluidic space by optically mediated electro-wetting (oEWOD); A first optical component, the first optical component being configured to form a first plurality of oEWOD traps for manipulating a plurality of microdroplets on the surface; A second optical component, the second optical component being configured to form a second plurality of oEWOD traps on the surface to maintain the relative positions of a plurality of microdroplets during adjustment of the first optical component and / or during a loading operation; And An inspection component, the inspection component being configured to interrogate the contents of a plurality of microdroplets.

2. The device according to claim 1, wherein The inspection component is an electromagnetic radiation source and is multiplexed with electromagnetic radiation from the first optical component.

3. The device according to claim 1 or claim 2, wherein, The first composite wall and the second composite wall are at least partially transparent, and the first optical component and the second optical component are located on opposite sides of the microfluidic space.

4. The device according to claim 1 or claim 3, wherein At least one of the first composite wall and the second composite wall is transparent, the first optical component and the second optical component are located on the same side of the microfluidic space, and wherein a color filter is applied to the second optical component to prevent interference with the first optical component.

5. The apparatus according to any one of claims 2 to 4, wherein, At least one of the first optical component and the second optical component includes a microlens array.

6. An apparatus for manipulating microparticles, the apparatus comprising: A chip, the chip including a first transparent composite wall and a second transparent composite wall, the first transparent composite wall and the second transparent composite wall defining a holding space and being configured to manipulate microparticles on a surface defining the holding space; A first optical component, the first optical component being configured to direct an optical beam through the first composite wall onto the surface to form a first plurality of optical traps for manipulating a plurality of microparticles on the surface; A second optical component, the second optical component being configured to direct an optical beam through the second composite wall onto the surface to form a second plurality of optical traps on the surface to maintain the relative positions of the plurality of microparticles during adjustment of the first optical component and / or during a loading operation; And An inspection component, the inspection component being configured to interrogate the contents of the plurality of microparticles.

7. An apparatus for inspecting or manipulating an array of microdroplets, the apparatus comprising: A microfluidic chip, the microfluidic chip including a first composite wall and a second composite wall that define a microfluidic space, and the microfluidic chip being configured to manipulate microdroplets on a surface defining the microfluidic space by optically mediated electro-wetting (oEWOD); A first optical component; A second optical component, the second optical component including a light source that acts as a holding light source during adjustment of the first optical component; And An inspection component, the inspection component being physically separated from the optical components.

8. The apparatus according to claim 7, wherein The first optical component is a high-resolution optical component, and the second optical component is a low-resolution optical component.

9. The device according to claim 7 or claim 8, wherein The first optical component and the second optical component are located on opposite sides of the microfluidic space.

10. The device according to any one of claims 7 to 9, wherein, The first composite wall and the second composite wall are substantially transparent.

Images