Methods and apparatus for high-throughput droplet manipulation

Abstract

The present invention provides methods and apparatus for manipulating and interrogating the contents of a large number of droplets on the surface of a microfluidic chip in parallel. According to one aspect of the present invention, a method for manipulating and interrogating droplets on a microfluidic chip by optically mediated electrowetting (oEWOD) is provided, the method comprising: forming a plurality of oEWOD traps on the 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 component while holding one or more droplets in place by the second array of oEWOD traps. An apparatus comprising a microfluidic chip and the first and second optical components is also provided.

Description

Technical Field

[0001] The present disclosure relates to methods and apparatus for manipulating droplets, and in particular to applying optically mediated electrowetting-on-device / optical electrowetting-on-dielectric (oEWOD) technology to manipulate and interrogate the contents of a large number of droplets on the surface of a microfluidic chip in parallel. Background Art

[0002] Electrowetting on dielectric (EWOD) is a well-known effect in which an electric field applied between a liquid and a substrate causes the liquid to wet more on the surface than it would naturally. The effect of electrowetting can be used to manipulate a fluid (e.g., move, divide, or change the shape of the fluid) by applying a sequence of spatially varying electric fields on a substrate to increase surface wettability following a sequence of spatial variations. 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.

[0003] In our published application WO2018 / 234445 (the entire contents of which are incorporated herein by reference), we have described an apparatus for manipulating droplets, which uses photoelectrowetting to provide the motive force. In this optically mediated electrowetting (oEWOD) apparatus, a droplet is 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 comprises a containment wall, hereinafter referred to as a "virtual" electrowetting electrode location, which is generated by selectively illuminating an area of ​​a semiconductor layer buried therein. By selectively illuminating the layer with light from a separate light source controlled by an optical assembly, a virtual path of virtual electrowetting electrode locations can be generated instantaneously, along which the droplet can be moved. Thus, the conductive unit is omitted and permanent droplet receiving locations are abandoned in favor of a uniform dielectric surface, on which droplet receiving locations are transiently generated by selectively and variably illuminating points on the photoconductive layer using, for example, a pixelated light source. This enables a highly localized electrowetting field to move the droplets over the surface by establishing induced capillary-type forces anywhere on the dielectric layer; optionally associated with any directional microfluidic flow of a carrier medium in which the droplets are dispersed, for example by emulsification.

[0004] Another disclosure of oEWOD is the single-sided open configuration platform “Single-sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns” by Park, Sung-Yong, Michael A. Teitell, and Eric PY Chiou in Lab on a Chip 10.13 (2010), pp. 1655-1661.

[0005] While these existing platforms allow fine-grained control of droplet motion, there is a practical limit (several thousand) to the number of droplets 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 typically required in the pharmaceutical industry, it is necessary to process droplet numbers on the order of 106 or more.

[0006] For example, in the fields of cell line development and antibody development, there is a need to allow initial screening of large numbers of biological agents (up to millions) to enable the number of agents to be reduced to a sensible number (thousands). To achieve an efficient workflow, this initial screening needs to be performed in a multiplexed manner on large numbers of biological agents. Summary of the invention

[0007] The present disclosure provides methods and related devices in which the flexibility of an oEWOD microfluidic chip is combined with two individually controllable optical components, both of which are capable of producing a fixed but switchable array of light spots on the surface of the oEWOD chip. Such a configuration enables high throughput and flexible loading and processing of droplets, thereby addressing the need for multiplexed processing in screening applications.

[0008] According to the present invention, a method for inspecting and / or selecting droplets on a microfluidic chip by optically mediated electrowetting (oEWOD) is provided, the method comprising: temporarily forming a plurality of oEWOD traps on the surface of the chip so that the plurality of droplets on the chip surface form an array of droplets; and maintaining the entire array of droplets while inspecting at least a subset of the array.

[0009] A typical implementation of the method of the present invention is cyclical and hierarchical. At a minimum, the method must involve either inspection of droplets or selection of a subset of droplets. Selection without inspection may be appropriate where there is uniformity across a subset of droplets and the subset can therefore be automatically selected without inspection. Inspection without selection is suitable for monitoring the state of multiple droplets. A typical mode of operation would be to inspect droplets and then select a subset of the inspected droplets based on 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 at the time of inspection.

[0010] Deploying oEWOD traps to create transient arrays offers considerable advantages over related techniques that rely on physical structures for housing droplets. The transient structures introduce considerable flexibility to create different structures and modify the structures in real time.

[0011] The term oEWOD trap used herein may also be referred to as a sprite. It is light projected onto a surface and it does not contain fences or wells permanently located on the surface. The fragments can form an array, move a portion of the array and rebuild a different array at a later time. Therefore, the surface on which the fragments are projected is effectively a blank canvas, not constrained by a permanent physical geometry for positioning multiple droplets.

[0012] The ability to hold the entire array of droplets while inspecting a subset of the array allows for sequential, detailed inspection of the array droplet-wise, down to the droplet without losing contact with the entire array. By inspecting a subset of the array, optical assemblies with different fields of view can be deployed without losing droplets that are not within the inspection field of view.

[0013] The step of maintaining the entire array of droplets may include maintaining the entire array in a stationary configuration. Alternatively, the maintaining step may include maintaining some or all of the array in motion. Some or all of the array may be maintained while traveling across the surface of the chip. The traveling may be at a substantially constant speed, or it may include deceleration when some or all of the droplets reach a stationary configuration in which they are subsequently maintained. This step may include capturing droplets that are not attached or locked to debris or OEWOD traps.

[0014] The step of maintaining the entire array of droplets may 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 OEWOD traps of the first array.

[0015] The alignment of the first and second arrays of oEWOD traps enables switching between the first and second optical assemblies forming the corresponding arrays, such that each optical assembly can inspect or manipulate a subset of the array of droplets while the entire array is held in place. In some embodiments, only the second optical assembly can hold the entire array, while both optical assemblies can inspect and hold a subset of the array.

[0016] Because either assembly can hold, inspect and manipulate at least a subset of the array, the responsibility for holding the droplets can be switched between the two assemblies in order to optimize the optical performance of the optical assembly responsible for detailed inspection and / or manipulation of a subset of the droplets.

[0017] Typically, one of the optical assemblies has a smaller field of view than the other, and is therefore capable of holding, inspecting and manipulating only a subset of the array. The optical assembly with the smaller field of view will be able to provide finer manipulation of droplets within the selected subset due to improved resolution of light debris.

[0018] The step of temporarily forming a plurality of OEWOD traps may be performed by an optical component, and the step of temporarily forming a second array of oEWOD traps on the surface may be performed by a second optical component.

[0019] The step of aligning one or more OEWOD traps of the second array with the OEWOD traps of the first array may enable the step of holding the entire array of droplets switchably between the first optical assembly and the second optical assembly.

[0020] In this context, the phrase entire array refers to all droplets currently being held, inspected, and / or manipulated. These droplets may be a uniform rectilinear array occupying the entire surface of the chip. However, when droplets are inspected, merged, separated, and otherwise manipulated, the array may cover only a portion of the chip. Furthermore, the droplets may not be a rectilinear array, but may be patterned. Furthermore, the phrase entire array refers to all droplets active at that time, such that if a subset of droplets is deselected and removed, the remaining droplets are the entire array at a subsequent time.

[0021] In addition, according to one aspect of the present invention, a method for manipulating and inspecting droplets on a microfluidic chip by optically mediated electrowetting (oEWOD) is provided, the method comprising: using a first optical component to form a plurality of oEWOD traps on the surface of the chip so that the plurality of droplets on the surface of the chip form an array of droplets corresponding to a first array of oEWOD traps; using a second optical component to form 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 droplets; and adjusting the first optical component while one or more droplets are held in place by the second array of oEWOD traps.

[0022] Having both a first optical component and a second optical component capable of forming an array of OEWOD traps for holding and manipulating droplets on the surface of the chip greatly enhances the operational flexibility of the microfluidic chip because one of these optical components can be used to hold all or a selected portion of a droplet in place on the surface while the other component is deactivated or adjusted, meaning that thousands of droplets can be manipulated using different parameters or moved to different locations without losing droplets during the interruption required to adjust one of these components.

[0023] In some embodiments, the method further includes: selecting a subset of droplets from the array of droplets based on an inspection of the contents of the droplets; deactivating all oEWOD traps except those oEWOD traps that captured the selected subset of droplets; and performing a flushing operation to remove droplets from the array of droplets that are not in the selected subset.

[0024] The steps of deactivating the oEWOD trap for unselected droplets (e.g., those droplets determined to be undesirable during a sorting operation) and performing a flushing operation to remove the undesired droplets 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 droplets can be an inspection to determine which droplets are empty and which contain cells to be subjected to further observation, and the unselected droplets to be flushed can be those that do not contain cells. In some embodiments, the flushing operation includes reordering the array of droplets using the oEWOD trap of the first optical assembly so that the removal of droplets that are not in the subset is not hindered by droplets in the subset, and / or allowing the continuous phase to enter the microfluidic chip through multiple fluid inlets to remove droplets that are not in the selected subset when the associated oEWOD trap has been deactivated.

[0025] An initial step in the flushing operation to reorder the array ensures that the removal of unwanted droplets is not impeded by droplets in the selected subset, and in particular that unwanted droplets do not collide with droplets that are marked for further inspection during removal. Such reordering can include, for example, switching unwanted droplets in the center of the array with those droplets on the outer edge of the array that are selected for further inspection. In addition, the use of a continuous phase to flush out unselected droplets further reduces the need for fine-grained control of droplets in large-scale operations, because the unselected droplets do not need to be manipulated across the surface of the chip using optical components, but rather simply maintain the position of the droplet in the selected subset by not deactivating the corresponding oEWOD trap. The continuous phase can be composed of any of silicone oil, mineral oil, and fluorocarbon oil.

[0026] In some embodiments, the adjustment to the first optical assembly includes at least one of a change in resolution, a change in magnification, a change in field of view, a change in a color selection element included in the assembly, and replacing a lens assembly closest to the sample being imaged. In some embodiments, the method further includes: performing a further inspection of the contents of the array of droplets using the first optical assembly after making the adjustment.

[0027] As described above, the methods of the present invention enable greater operational flexibility in droplet assays, particularly for large-scale droplet manipulation. In some embodiments, the methods of the present invention enable adjustment of the parameters of the assay without losing any droplets from the initial array formed. For example, a first assay can be performed using a first optical assembly with a wide field of view on an array comprising thousands of droplets, a subset of those droplets can be selected for further inspection, and then a second optical assembly can hold the selected droplets in place while reducing the field of view of the first optical assembly to allow more precise droplet manipulation or inspection. This example is not limiting, and the principle of a selected subset or all of the droplets in an array of droplets being held in place by a second optical assembly while adjusting the parameters of the first optical assembly between consecutive assays can be applied, thereby improving efficiency and fine-tuning droplet assays in various ways.

[0028] In some embodiments, a first optical assembly according to the present invention as disclosed herein may be used to move, merge and / or split the droplets.

[0029] In some embodiments, a second optical assembly according to the invention disclosed herein may be used to manipulate droplets, such as to move, merge and / or split the droplets on a microfluidic chip by optically mediated electrowetting (oEWOD).

[0030] In some embodiments, the method further includes deactivating the first optical component and translating the array of droplets across the surface of the microfluidic chip using an oEWOD trap formed by a second optical component. Microfluidic chips typically include a plurality of different areas that are designed to perform specific operations therein. For example, the surface of the droplet may include a classification area, an inspection area, or an area where the surface of the chip has been processed to be suitable for assays using a specific type of cell. Another way to improve the operational flexibility of such assays may be by using a dual component configuration in which the same array of droplets or a selected subset thereof is transported between such areas by a second optical component before or after performing the assay. Using a second optical component to translate the entire array of oEWOD traps has the following advantages: fine droplets that are not required for droplet control by the oEWOD traps, and the relative positions of the droplets in the transported array are maintained relative to each other.

[0031] In some embodiments, the first optical assembly has a higher imaging resolution than the second optical assembly. In a dual assembly configuration of the present invention, it may be advantageous to have one optical assembly designated as the creator of a "holding array" capable of holding and delivering a large number of droplets at a time, and the other optical assembly responsible for a high-resolution, adjustable array suitable for achieving fine-grained control of the droplets in the array when necessary.

[0032] In some embodiments, the step of forming an array of droplets includes the following initial steps: forming a plurality of oEWOD traps in the shape of a target array using a second optical component; determining the position of the plurality of droplets on the surface of the microfluidic chip using a first optical component; and manipulating the plurality of droplets 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 for some chip configurations.

[0033] In other embodiments, the step of forming an array of droplets includes: forming a first array of oEWOD traps using a first optical assembly; and loading a plurality of droplets onto a surface of a chip where the first array of oEWOD traps is located. Forming an array of oEWOD traps on a surface of a chip such that droplets located on the surface of the chip coalesce onto the oEWOD trap array locations is an effective method of forming an array of droplets in assays involving large numbers of droplets to be analyzed because this method eliminates the need for precise droplet control by a droplet during a loading phase. In this manner, arrays of thousands of droplets can be quickly and easily formed.

[0034] In some embodiments, electromagnetic radiation from the first optical assembly is multiplexed with electromagnetic radiation from an inspection component configured to inspect the droplet and its contents. Combining the inspection component with the optical assembly used to form the oEWOD trap allows for more efficient directing of the inspection radiation.

[0035] In some embodiments, the inspection of the contents of the droplet 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 indicator is local optical plasmon resonance on metal nanoparticles, the particles are functionalized with antigens or antibodies, and the detection method detects changes in the spectral response of the functionalized nanoparticles in response to the binding of the target molecule to the surface.

[0036] In some embodiments, at least one of the first array and the second array is formed using a projection optical device consisting 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.

[0037] According to another aspect of the present invention, a device for manipulating droplets is provided, comprising: a microfluidic chip, the microfluidic chip comprising a first composite wall and a second composite wall defining a microfluidic space, and the microfluidic chip being configured to manipulate droplets on a surface defining the microfluidic space by optically mediated electrowetting (oEWOD); a first optical component, the first optical component being configured to form a first plurality of oEWOD traps to manipulate a plurality of droplets on a surface; a second optical component, the second optical component being configured to form a second plurality of oEWOD traps on the surface to maintain relative positions of a plurality of droplets during adjustment of the first optical component and / or during a loading operation; and an inspection component configured to interrogate the contents of a plurality of droplets.

[0038] In some embodiments, the inspection component is a source of electromagnetic radiation and is multiplexed with the electromagnetic radiation from the first optical assembly.

[0039] 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 wherein a color filter is applied to the second optical component to prevent interference with the first optical component.

[0040] In some embodiments, at least one of the first optical component and the second optical component comprises a microlens array. In some embodiments, the second optical component or both optical components have relatively coarse-grained optical control; not necessarily switching each illumination spot independently, but arranging them into small groups that can be actuated individually.

[0041] In some embodiments, the apparatus further comprises an arrangement of multiple sets of external flow control valves and pumps and inlets for loading and flushing operations.

[0042] In some embodiments, the optical assembly is configured to provide an array of spots having a diameter between 20µm and 250µm and formed at a pitch between 50µm and 675µm, in particular an array of spots having a diameter between 30µm and 250µm and formed at a pitch between 30µm and 300µm. The pitch is typically 2.5 times the droplet diameter. In some embodiments, the optical assembly is configured to provide an array of spots having an approximate pitch of 100µm or 125µm and a spot size of approximately 50µm. In some embodiments, the optical assembly is configured to provide an array of spots having a diameter between 5µm and 30µm and formed at a pitch between 12.5µm and 75µm.

[0043] In some embodiments, the methods of the present invention are applied to optically activated devices, such as devices configured to manipulate microparticles (including microparticles in droplets) by dielectrophoresis. Cells or particles are manipulated and examined using functionally equivalent optical instruments to generate virtual optical dielectrophoretic gradients. Microparticles as defined herein may refer to particles, such as biological cells, microbeads made of materials including polystyrene and latex, magnetic microbeads, or colloids.

[0044] Similar to the method described above for optical electrowetting, a first high-resolution optical component is used to perform fine manipulation and detailed inspection of particles and / or cells by a combination of optically mediated dielectrophoresis. A coarse 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 coarse optical component to hold and transport very large numbers of particles and / or cells.

[0045] Therefore, according to another aspect of the present invention, a device for manipulating particles is provided, the device comprising: a chip comprising 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 particles located on a surface defining the holding space; a first optical component, the first optical component being configured to guide an optical beam onto a surface through the first composite wall to form a first plurality of optical traps to manipulate a plurality of particles on the surface; a second optical component, the second optical component being configured to guide an optical beam onto a surface through the second composite wall to form a second plurality of optical traps on the surface to maintain the relative positions of the plurality of particles during 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 particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 shows a cross-sectional view of an example microfluidic chip as described in WO2018 / 234445;

[0047] Figure 2 shows a cross-sectional view of an exemplary microfluidic chip suitable for performing the method of the present invention;

[0048] Figure 3 shows a top view of the surface of the microfluidic space;

[0049] FIG. 4A to FIG. 4E An example of the process of a loading operation and a droplet query workflow is shown;

[0050] Figure 5A and Figure 5B An illustration of a merging operation with droplets according to the present invention is provided;

[0051] Figure 5C and Figure 5D Provides an illustration of a droplet splitting operation according to the present invention;

[0052] Fig. 6A and Figure 6B An alternative illustration of a merging operation with droplets according to the present invention is provided; and

[0053] Figure 6C and Fig.6D An alternative illustration of the splitting operation of a droplet according to the present invention is provided. DETAILED DESCRIPTION

[0054] In order 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.

[0055] refer to Figure 1, showing a cross-sectional view of an exemplary microfluidic chip device (as described in WO2018 / 234445) comprising an oEWOD structure suitable for rapid manipulation of aqueous droplets.

[0056] The device includes a top glass plate 13 and a bottom glass plate 14, each 500 µm thick and coated with a transparent conductive indium tin oxide (ITO) layer 15 having a thickness of 130 nm. Each ITO layer 15 is connected to an A / C source 16, with the ITO layer on the bottom glass plate 14 being 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 aluminum oxide or hafnium dioxide layer 18, which in turn is coated with a silicon dioxide interstitial layer supporting a trichloro(1H,1H,2H,2H-perfluorooctyl)silane layer 19 to render the surface of the aluminum oxide / hafnium dioxide layer 18 hydrophobic. The top glass plate 13 and the amorphous silicon layer 17 are spaced 80 µm apart using spacers so that the droplets are somewhat compressed when introduced into the device.

[0057] An image of a reflective pixelated screen illuminated by a first optical assembly (in this example, an LED light source) 20 is typically disposed below the bottom glass plate 14 and visible light (wavelength 660nm or 830nm) at a level of 0.01Wcm-2 is emitted from each diode 21 and impinges on the amorphous silicon layer 17 by propagating in the direction of a plurality of upward arrows through the bottom layers 14 and 15. At each point of incidence, a photoexcited region 22 of charge is created in the amorphous silicon layer 17, which induces a modified liquid-solid contact angle in the aluminum oxide / hafnium dioxide layer 18 at the corresponding electrowetting locations 23 to create oEWOD traps at these locations. The modified nature of the oEWOD trap locations provides the capillary force required to hold the droplet 2 in place or propel the droplet 2 from one point 23 to another. The optical assembly 20 is controlled by a microprocessor 24, which determines which diodes 21 in the array are illuminated at any given time through a pre-programmed algorithm.

[0058] refer to Figure 2 , showing that Figure 1 2, wherein 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 processing a large number of droplets 2.

[0059] The light source from the second optical assembly can be activated and aligned to the location of a droplet held by the first light source during a loading option, and / or can act as a holding light source during adjustments to the first optical assembly, such as during switching of a lens in the first light source, during interrogation, or during translation of the droplet array across the surface of the microfluidic space.

[0060] The light source of the optical assembly need not 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 will be suitable. For example, the projection optics may consist of an LED or LCD screen combined with a microlens array arrangement or a Fly'Eye arrangement. In examples of the present disclosure, the illumination pattern of the photoelectrowetting is spatially modulated across the object plane (i.e., the plane of the surface on which the oEWOD traps are formed) using, for example, a digital micromirror device, an LCD display, a spatial light modulator, or an LED array. The example projected spot array may consist of points with a diameter of 50 um at a pitch of 100 um (i.e., the center-to-center distance between the spots).

[0061] In some examples, Figure 2 The device comprises a combined inspection and manipulation optical assembly in which light suitable for photoelectrowetting manipulation is multiplexed with light suitable for fluorescence excitation. Alternatively, the inspection and manipulation components may be physically separated, for example if the interrogation component is not a source of electromagnetic radiation but a passive collection system.

[0062] refer to Figure 3 , illustrates a top view of the surface of a microfluidic space that has been loaded with a large number of droplets. In this example, the field of view of the first optical component 20 (i.e., the area of ​​influence exerted on the droplets 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. Figure 3 In the example configuration of , it can be seen that the first optical component has a much narrower field of view and is therefore more suitable for fine manipulation of a smaller number of droplets, while the second optical component has a much wider field of view suitable for holding a large number of droplets in place across a large portion of the surface.

[0063] Typically, the inspection component has an objective lens having a high numerical aperture suitable for maximizing light collection efficiency and resolution of fluorescence imaging. By switching the objective lens, the imaging magnification can be increased or decreased and the resolution and collection efficiency can be increased or decreased concomitantly during the determination of the contents of the droplets in the inspection chip. Increasing the magnification may require reducing the field of view of the imaging system.

[0064] In cases where the inspection optics and the electro-electrowetting manipulation optics are multiplexed, a reduction in the field of view will cause droplets held in place by the manipulation pattern to become outside the field of view of the optics; during this time, they may move away by diffusion or fluid flow.

[0065] 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 the light from the manipulation pattern from interfering with the fluorescence image; during such a long interruption, the droplets may again move in an uncontrolled manner.

[0066] These limitations can be overcome by combining low-resolution spot generation optics with high-resolution inspection and manipulation components.

[0067] In an example process, a high-resolution optical assembly is used to position a droplet into a specific layout in a microfluidic chip, and then the droplet is imaged. A pattern aligned with the droplet position is then generated on a low-resolution spot generation assembly. This pattern from the low-resolution assembly can then be activated as a holding pattern when the objective lens of the inspection assembly is changed; the pattern can also be used to hold droplets that end up outside the field of view, and the pattern can be used to hold droplets during fluorescence acquisition. Alternatively, in some embodiments, droplets can be positioned to a specific layout by a low-resolution optical assembly and inspected by a high-resolution optical assembly.

[0068] The process can be controlled by software generating a pixel map on the target surface from the two illumination sources 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 ranges used for the high-resolution source.

[0069] If a broadband light source is chosen for the low-resolution optical assembly, interference with the light used for fluorescence imaging can be eliminated by applying a blocking "notch" filter to the input light, which removes a band of the spectrum while allowing light outside of that band to be used to preserve the pattern.

[0070] In addition, the low-resolution optical assembly can be used as a holding mechanism to maintain the relative position between the droplets as the sample is moved. For example, if a mechanical motorized motion stage is needed to translate the sample, the pattern registration can be moved so that the droplets move in almost unison with the stage in a stepwise manner so that their relative positions remain unchanged.

[0071] In order to overlap the two photoelectrowetting control patterns (from the high-resolution component and the low-resolution component), it is preferred to use a substantially transparent photoelectrowetting device so that one 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 so that the light enters outside the numerical aperture of the objective lens of the high-resolution component. In this case, compensating optical elements are preferably added to the low-resolution projector to adjust the shape and focus of the projected pattern to avoid image deformation caused by projecting at an oblique angle.

[0072] refer to FIG. 4A to FIG. 4E , shows another example process of loading operation and droplet query workflow, which is suitable for processing a large number of droplets, such as more than one million particles, without requiring precise droplet-by-droplet control.

[0073] refer to Figure 4A , in a first step, a light spot projected by an optical component in the shape of an array causes photoexcitation of the photoactive layer, resulting in charge accumulation on the surface of the dielectric layer, which serves as an optical trap for microdroplets on the surface of the microfluidic space within the microfluidic chip (oEWOD trap).

[0074] See also Figure 4B In a second step, droplets of biological agents (e.g., cells) are introduced into the device under flow in a continuous phase that may consist of oil (e.g., silicone oil, mineral oil, or fluorocarbon oil). The interaction between the droplets containing the biological agent and the optical trap causes the droplets to self-arrange and merge into a pattern that matches the array pattern of the projection optics (optical assembly).

[0075] refer to Figure 4C The array of captured droplets is then interrogated through a transparent substrate of the microfluidic chip, wherein the results of the interrogation are used to select a subset of droplets based on a predetermined criterion, such as fluorescence imaging or surface plasmon resonance detection on metal nanoparticles.

[0076] refer to Figure 4D , when a subset of droplets has been selected, light spots corresponding to unwanted droplets that are not in the selected subset are turned off and the corresponding droplets are removed from the interrogation area of ​​the surface of the chip, optionally completely removed from the microfluidic chip under a continuous flow introduced into the microfluidic chip by a series of pumps and valves for this purpose.

[0077] refer to Figure 4E The selected set of droplets then either flows off the chip for further post-processing or analysis, or is retained on the chip for further processing and analysis using the previously described arrangement using a micromirror array to control the light spot and droplet motion.

[0078] Apart from Figure 1 In addition to the specific examples provided and illustrated, general characteristics and optional features of oEWOD microfluidic chips suitable for performing the methods of the present invention, and the composite structures included therein, will now be described.

[0079] The oEWOD structure comprises: a first composite wall, the first composite wall consisting 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 having a wavelength in the 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, the second composite wall consisting 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 first dielectric layer and a second dielectric layer are provided with 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 a microdroplet; an A / C source for providing a voltage between a first composite wall and a second composite wall connecting the first conductor layer and the second conductor layer; a first electromagnetic radiation source and a second electromagnetic radiation source, wherein the first electromagnetic radiation source and the second electromagnetic radiation source have an energy higher than the band gap of the photoactive layer suitable for incident on the photoactive layer to induce corresponding virtual electrowetting sites on the surface of the first dielectric layer; and a device for manipulating the incident point of the electromagnetic radiation on the photoactive layer to change the arrangement of the virtual electrowetting sites to create at least one electrowetting path along which the microdroplet can be moved. The first wall and the second wall of these structures are transparent, wherein the microfluidic space is sandwiched between the first wall and the second wall.

[0080] Suitably, the first substrate and the second substrate are made of mechanically strong materials, such as glass metal or engineered plastics. In some embodiments, the substrates may 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.

[0081] The first conductor layer and the second conductor layer are located on one surface of the first substrate and the second substrate, and typically 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. 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, wherein electromagnetic radiation is guided between the gaps of the grid.

[0082] The photoactive layer suitably comprises a semiconductor material that can generate local charge areas in response to stimulation by a second electromagnetic radiation source. Examples include hydrogenated amorphous silicon layers 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 with a thickness ranging from 30 nm to 160 nm. The dielectric properties of this layer preferably include a high dielectric strength of> 10^7V / 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 aluminum oxide, silicon dioxide, hafnium oxide or a thin non-conductive polymer film.

[0083] In another embodiment of these structures, at least the first dielectric layer (preferably both) is coated with an anti-fouling layer to help establish the desired droplet / carrier fluid / surface contact angles at the various virtual electrowetting electrode locations and additionally 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, the second anti-fouling layer can be applied directly to the second conductor layer.

[0084] For best performance, the antifouling layer should help establish a droplet / carrier fluid / surface contact angle that should be in the range of 50 to 180° when measured at 25°C as an air-liquid-surface three-point interface. In some embodiments, these layers have a thickness of less than 10 nm and are typically monolayers. In another embodiment, these layers comprise polymers of acrylates such as methyl methacrylate or derivatives thereof substituted with hydrophilic groups (e.g., alkoxysilyl groups). One or both of the antifouling layers are hydrophobic to ensure best performance. In some embodiments, a gap layer of silicon dioxide less than 20 nm in thickness may be placed between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0085] 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 in which the droplets are contained. Preferably, before the droplets are contained, the droplets themselves have an intrinsic 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 droplets are subjected to compression, thereby resulting in enhanced electrowetting performance, for example, through better droplet merging capabilities. In some embodiments, the first dielectric layer and the second dielectric layer are coated with a hydrophobic coating, such as fluorosilane.

[0086] 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 spacers include beads or columns, ridges produced by an intermediate resist layer that has been produced by photopatterning. Alternatively, a deposited material (such as silicon oxide or silicon nitride) can be used to create a spacer. Alternatively, a thin film layer (including a flexible plastic film with or without an adhesive coating) can be used to form a spacer layer. Various spacer geometries can be used to form narrow channels, tapered channels, or partially enclosed channels defined by rows of columns. Through careful design, these spacers can be used to help the deformation of droplets, and then the deformed droplets are subjected to droplet splitting and effect operations. Similarly, when loading the chip under hydraulic pressure, these spacers can be used to physically separate the various areas of the chip to prevent cross-contamination between droplet populations and promote droplets to flow in the correct direction.

[0087] The first and second walls are biased using an AC power supply 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 source of electromagnetic radiation having a wavelength in the range of 400nm to 850nm (preferably 660nm) and an energy exceeding 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.

[0088] Where the electromagnetic radiation source is pixelated, the electromagnetic radiation is supplied directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated by light from an LED or other lamp. This enables highly complex patterns of virtual electrowetting electrode locations to be rapidly created and destroyed on the first dielectric layer, thereby enabling droplets to be precisely steered along essentially any virtual path using tightly controlled electrowetting forces. Such an electrowetting path can be viewed as consisting of a continuum of virtual electrowetting electrode locations on the first dielectric layer.

[0089] The points of incidence of the electromagnetic radiation source on the photoactive layer may 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, the morphology of these points corresponds in whole or in part to the morphology of the droplet once it enters the microfluidic space. In one embodiment, the points of incidence and therefore the electrowetting electrode locations may be crescent-shaped and oriented in the expected direction of travel of the droplet. Suitably, the electrowetting electrode locations themselves are smaller than the surface of the droplet adhered to the first wall and give the maximum field intensity gradient at the contact line formed between the droplet and the surface dielectric.

[0090] In some embodiments of the oEWOD structure, the second wall further comprises a photoactive layer that enables induction of virtual electrowetting electrode locations on the second dielectric layer by the same or different electromagnetic radiation source. Adding the second dielectric layer enables the wetting edge of the droplet to transition from the upper surface to the lower surface of the structure and applies more electrowetting forces to each droplet.

[0091] The first dielectric layer and the second dielectric layer may be composed of a single dielectric material, or it may be a composite of two or more dielectric materials. The dielectric layer may be made of, but not limited to, Al2O3 and SiO2.

[0092] A structure may be provided between the first dielectric layer and the second dielectric layer. The structure between the first dielectric layer and the second dielectric layer may be made of, but not limited to, epoxy, polymer, silicon or glass or a mixture or composite thereof, with straight, inclined, curved or microstructured walls / surfaces. The structure between the first dielectric layer and the second dielectric layer may be connected to the top composite wall and the bottom composite wall 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. Alternatively or in addition, the conductors and dielectrics may be deposited on a shaped substrate that already has walls.

[0093] Some aspects of the methods and apparatus of the present invention are suitable for application to optically activated devices other than electrowetting devices, such as devices configured to manipulate microparticles by dielectrophoresis or optical tweezers. In such devices, cells or particles are manipulated and examined using functionally identical optical instruments to produce virtual optical dielectrophoretic gradients. Microparticles as defined herein may refer to microparticles such as biological cells, microbeads made of materials including polystyrene and latex, hydrogels, magnetic microbeads, or colloids. Dielectrophoresis and optical tweezers mechanisms are well known in the art and can be readily implemented by those skilled in the art.

[0094] Similar to the method described above for optical electrowetting, a first high-resolution optical component is used to perform fine manipulation and detailed inspection of particles and / or cells by a combination of optically mediated dielectrophoresis. A coarse 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 coarse optical component to hold and transport very large numbers of particles and / or cells.

[0095] refer to Figure 5A and Figure 5B , provides an illustration of a merging operation using a second optical arrangement as described herein to hold droplets within a large area 52 and using a first optical arrangement as disclosed herein to merge droplets within a smaller field of view 54. Figure 5A Figure 2 shows the droplets before merging. Figure 5AThe arrows shown illustrate the direction in which the droplets are merged together. Figure 5B The merged droplet is shown after the merging operation.

[0096] refer to Figure 5C and Figure 5D , provides an illustration of a splitting operation using a second optical arrangement as described herein to hold a droplet in a large area 52 and using a first optical arrangement as described herein to split the droplet in a smaller field of view 54. Figure 5C The arrows shown illustrate the direction in which a droplet breaks off to provide another droplet. Figure 5D Post-split events following a split operation are shown.

[0097] refer to Fig. 6A and Figure 6B , provides an illustration of a merging operation in which droplets are maintained between operations of the second optical device 52 and merged during operations using the second optical device 52 as disclosed herein. Fig. 6A The arrows indicated in illustrate the direction of the droplets during the merging operation. Figure 6B The merged droplet is shown after the merging operation.

[0098] refer to Figure 6C and Fig.6D , provides an illustration of a splitting operation of a droplet held between operations of the second optical assembly 52, and splitting a droplet during a splitting operation using the second optical assembly. Figure 6C The arrows indicated in show the direction in which a droplet breaks apart to form additional droplets during a breaking operation. Fig.6D A plurality of droplets are shown formed after the splitting operation.

[0099] The optical assembly that inspects a subset of the array has a much smaller field of view than the optical assembly that holds the array in place. Within the reduced field of view, there may be only one droplet. Alternatively, there may be 24, 48, 256, 1048, or any suitable number of droplets in the field of view of the inspection optical assembly. Inspection may be performed droplet by droplet, with the optical assembly scanning through its field of view to sequentially inspect each droplet. This may involve the optics being in a single position, and the scanning involving inspecting a portion of the FOV by processing information from a portion of an image that falls on an imaging sensor (e.g., a camera that forms part of the optical assembly). Alternatively or in addition, the optical assembly may be integrated over its entire field of view to obtain an overview of the proportion of droplet emission. This coarse-grained data may be combined with the droplets by droplet review in order to quickly focus on the most information-rich portion of the array.

[0100] Various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0101] As used herein, "and / or" should be considered as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0102] Unless the context dictates otherwise, the descriptions and definitions of the features above are not limited to any specific aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0103] It will be further appreciated by those skilled in the art that, while the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

1. A method for inspecting and / or selecting droplets 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 using a second optical component so that the plurality of droplets on the surface of the chip form an array of droplets; maintaining the entire array of droplets while inspecting at least a subset of the array, Wherein the step of inspecting at least a subset of the array is facilitated by the following sub-steps: The first optical assembly is adjusted while holding the one or more droplets in place.

2. The method of claim 1, wherein the step of maintaining the entire array of droplets is facilitated by the following sub-steps: temporarily forming a second array of oEWOD traps on the surface of the chip; and One or more oEWOD traps of the second array are aligned with the oEWOD traps of the first array.

3. The method according to claim 1 or claim 2, wherein: The step of temporarily forming the plurality of oEWOD traps is performed by an optical component, and the step of temporarily forming a second array of oEWOD traps on the surface is performed by a second optical component.

4. The method according to claim 2, wherein: the step of temporarily forming the plurality of oEWOD traps is performed by an optical component, and the step of temporarily forming a second array of oEWOD traps on the surface is performed by a second optical component, Therein, the step of aligning one or more oEWODs of the second array with the oEWOD traps of the first array enables the step of switching the holding of the entire array of droplets between the first optical assembly and the second optical assembly.

5. The method according to claim 1 or 2, wherein: The method further comprises manipulating the droplet in the following steps: selecting a subset of droplets from the array of droplets based on an inspection of the contents of the droplets; deactivating all oEWOD traps except those that capture the selected subset of droplets; as well as A flushing operation is performed to remove droplets from the array of droplets that are not in the selected subset.

6. The method according to claim 3, wherein: The method further comprises manipulating the droplet in the following steps: selecting a subset of droplets from the array of droplets based on an inspection of the contents of the droplets; deactivating all oEWOD traps except those that capture the selected subset of droplets; as well as performing a flushing operation to remove droplets from the array of droplets that are not in the selected subset, Wherein, the flushing operation includes: reordering the array of droplets using the oEWOD trap of the first optical assembly or the second optical assembly such that removal of droplets not in the subset is not hindered by droplets in the subset, and / or When the associated oEWOD trap has been deactivated, the continuous phase is admitted into the microfluidic chip through a plurality of fluid inlets to remove droplets that are not in the selected subset.

7. The method according to claim 1, wherein: The adjustment of the first optical component includes at least one of: a change in resolution, a change in magnification, a change in field of view, a change in illumination source, a change in a color selection element included in the component, and replacement of a lens component closest to a sample being imaged.

8. The method according to claim 1, wherein the method further comprises: After making the adjustments, further inspection of the contents of the array of droplets is performed using the first optical assembly.

9. The method according to claim 3, wherein: The method further comprises: The first optical assembly is deactivated, and the array of droplets is translated across the surface of the microfluidic chip using the oEWOD trap formed by the second optical assembly.

10. The method according to claim 3, wherein: The steps of forming the array of droplets include the following initial steps: forming a plurality of oEWOD traps in the shape of a target array using the second optical assembly; determining the positions of the plurality of droplets on the surface of the microfluidic chip using the first optical assembly; The plurality of droplets are manipulated into an array matching the target array of oEWOD traps using the plurality of oEWOD traps formed by the first optical assembly.

11. The method according to claim 3, wherein: The steps of forming the array of droplets include: forming a first array of oEWOD traps using the first optical assembly; and The plurality of droplets are loaded onto the surface of the chip where the first array of oEWOD traps is located.

12. The method according to claim 1 or claim 2, wherein: Inspection of the contents of the droplet is performed using at least one of: fluorescence imaging, localized optical plasmon resonance on metal nanoparticles, FRET, dark field, bright field, Raman, absorption, quantum dot fluorescence, spectroscopy.

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