Methods and apparatuses for high throughput microdroplet manipulation

Abstract

To provide methods and apparatuses for manipulating and interrogating the contents of large numbers of microdroplets in parallel on a surface of a microfluidic chip.SOLUTION: A method for manipulating and inspecting microdroplets on a microfluidic chip by optically mediated electrowetting (oEWOD) is provided which comprises: forming, using a first optical assembly, a plurality of oEWOD traps on a surface of the chip; forming, using a second optical assembly, a second array of oEWOD traps on the surface of the chip; and making an adjustment to the first optical assembly while one or more of the microdroplets are held in place by the second array of oEWOD traps. An apparatus comprising a microfluidic chip and first and second optical assemblies is also provided.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a method and apparatus for manipulating microdroplets, particularly for manipulating multiple droplets on the surface of a microfluidic chip. Optically mediated electrowetting for parallel manipulation and interrogation of microdroplet contents Application of Electrowetting-on-Device / Optical Electrowetting-on-Dielectric (oEWOD) Technology Regarding use. [Background technology]

[0002] Electrowetting on dielectric (EWOD) is a technique that uses an applied voltage between a liquid and a substrate. This is a known effect where a magnetic field makes a liquid on a surface more wettable than it would be naturally. The effect of the ion beam is used to generate a fluid by applying a series of spatially varying electric fields to the substrate. can be manipulated (e.g., moved, split, or reshaped) to change the surface wetting according to a series of spatial changes. Droplets manipulated with electrowetting-based devices are typically sandwiched between two parallel plates and actuated by multiple digital electrodes. The size of the electrodes determines the minimum droplet size that can be manipulated and the rate and schedule at which droplets can be processed in parallel. Limit the rules.

[0003] No. 6,269,999, the entirety of which is incorporated herein by reference. A device for manipulating microdroplets using optical electrowetting to provide the driving force has been developed. This optically mediated electrowetting (oEWOD) device In the chair, the microdroplets are placed in a microfluidic space defined by a containing wall, e.g., a microfluidic It is transported through a pair of parallel plates with a space between them. At least one of the containment walls is embedded within These are referred to below as "virtual" elements, which are generated by selectively irradiating regions of the embedded semiconductor layer. The position of the electrode is controlled by an optical assembly. Selective illumination of the layer with light from a light source results in the virtual electrowetting electrode positions It is possible to temporarily generate a virtual path along which the droplets can be moved. Therefore, the conductive cell can be omitted and a permanent droplet receiving position can be omitted, e.g., a pixel The droplet receiving position is determined by selectively varying the illumination of a point on the photoconductive layer using a controlled light source. This uses a homogeneous dielectric surface where the surface is temporarily etched by induced capillary forces. A highly localized electrowetting field that can move the microdroplets on the In some cases, the microdroplets are dispersed in a carrier medium, for example by emulsification. It can be placed anywhere on the dielectric layer in relation to the fluid flow.

[0004] Another disclosure of oEWOD is the single-sided open configuration platform of Non-Patent Document 1.

[0005] These existing platforms allow for fine-grained analysis by handling samples using microscope optics. Although it enables the control of the movement of microdroplets, the number of droplets that can be processed in parallel within a single field of view is practically However, in some applications, especially in the pharmaceutical industry, For large-scale screening applications such as those required, 6 It is necessary to handle droplet numbers on the order of 100 or more. There is a need.

[0006] For example, in the areas of cell line and antibody development, large numbers of biological constructs (up to millions) are required. Initial screening of potential agents reduces the number of active agents to a significant number (several thousand). This initial script should be able to be executed in order to achieve an efficient workflow. The screening needs to be multiplexed across multiple biological agents. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 234445 [Non-patent literature]

[0008] [Non-Patent Document 1] Park, Sung-Yong, Michael A. Teitell, and Eric PY Chiou, "Single-sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns." Lab on a Chip 10.13 (2010): 1655-1661. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure aims to maximize the flexibility of the oEWOD microfluidic chip by providing a Two independently controllable laser diodes capable of generating a fixed but switchable array of light spots on a In combination with an optical assembly, a method and related apparatus are disclosed. This allows for high-throughput and flexible injection and processing of microdroplets, thus facilitating screening. This eliminates the need for multi-processing in programming applications.

[0010] According to the present invention, optically mediated electrowetting (oEWOD) A method for inspecting and / or selecting microdroplets on a microfluidic chip, comprising: Multiple oEWOD traps are formed on the chip to transfer microdroplets to multiple microdroplets on the surface of the chip. forming an array of the microorganisms; and inspecting at least a subset of the array. and retaining the entire array of droplets.

[0011] A typical implementation of the method of the present invention is cyclical and hierarchical. At a minimum, the method involves It must involve the examination of small droplets or the selection of a subset of microdroplets. There is uniformity across the set, so the subset can be selected automatically without testing. In this case, the no-inspection option may be applicable. The normal mode of operation is to inspect the microdroplets, then The goal is to select a subset of microdroplets to be inspected based on information collected from the sample. Once selected, a subset of the microdroplets can be retained and / or manipulated. The cycle of selection can then be repeated, based on the data collected during the test. This may be followed by a holding and / or manipulation step.

[0012] The development of oEWOD traps for creating temporary arrays offers a promising solution for accommodating microdroplets. This has a significant advantage over related technologies that rely on physical structures. This provides greater flexibility in configuration and real-time structure changes.

[0013] As used herein, the term oEWOD trap may also be referred to as a sprite. This is a light projection onto a surface, with a pen or well permanently positioned on the surface. No. Sprites form an array, move a percentage of the array, and The array can then be reshaped, thus changing the surface onto which the sprites are projected. In effect, the microdroplet is positioned on a blank canvas, unconstrained by any permanent physical geometry. It's a bus.

[0014] The ability to retain the entire array of microdroplets while examining a subset of the array allows for the entire array to be examined. The array can be inspected in detail, down to the individual droplets, without losing contact with the By inspecting a subset of the array, droplets that are not within the inspection field can be detected without losing any of them. Optical assemblies with different fields of view can be arranged.

[0015] The step of holding the entire array of microdroplets includes holding the entire array in a stationary configuration. Alternatively, the holding step may include movably holding part or all of the array. Part or all of the array may be held while being advanced across the surface of the chip. This advancement may be at a substantially constant rate, or some or all of the microdroplets may be moved at a constant rate. This step may involve deceleration until the splice reaches a rest configuration that is then held. This may include capturing droplets that are not attached or locked in the trap or oEWOD trap.

[0016] The step of holding the entire array of microdroplets is performed by placing the oEWOD trap on the surface of the chip. a substep of temporarily forming a second array and one of the oEWOD traps of the second array; or by a substep of aligning multiple oEWOD traps with the first array of oEWOD traps. It could be.

[0017] The alignment of the first and second arrays of EWOD traps allows the first array to form This allows for handoff between the first and second optical assemblies, holding the entire array in place. Each optical assembly inspects or manipulates a subset of the microdroplet array while In some embodiments, only the second optical assembly holds the entire array. While both optical assemblies inspect and hold a subset of the array, It is possible.

[0018] Each of the two assemblies holds, inspects, and manipulates at least a subset of the array. This allows for the optical assembly to be responsible for detailed inspection and / or manipulation of that subset of microdroplets. To optimize the optical performance of the device, the droplets are held between the two assemblies. It can be handed over.

[0019] Typically, one of the optical assemblies has a smaller field of view than the other, and therefore the sub-areas of the array Only the optical assembly with the smaller field of view can be held, inspected, and manipulated. The improved resolution of the optical sprites allows for finer manipulation of droplets within the selected subset. It is possible to perform the work.

[0020] The step of temporarily forming the plurality of oEWOD traps is performed by an optical assembly. and temporarily forming a second array of oEWOD traps on the surface. can be performed by a second optical assembly.

[0021] One or more of the oEWOD traps in the second array are connected to the oEWOD traps in the first array. The step of aligning includes distributing a microdroplet between the first optical assembly and the second optical assembly. This may allow for the step of handing over the entire array of data.

[0022] In this context, the phrase "entire array" refers to the microdroplets currently being held, inspected, and / or manipulated. These droplets can be in a uniform linear array that covers the entire surface of the chip. However, as the droplets are inspected, combined, split, and otherwise manipulated, The array need only cover a portion of the chip. Furthermore, the droplets do not have to be in a linear array. Furthermore, the phrase "the entire array" refers to the entire array currently being executed. It refers to the entire microdroplet, so if a subset of the microdroplets is deselected and removed, the remaining The remaining droplets are the entire array at a later point in time.

[0023] Furthermore, in accordance with one aspect of the present invention, optically mediated electrowetting (o A method for manipulating and inspecting microdroplets on a microfluidic chip by EWOD, comprising: 1. Form multiple oEWOD traps on the surface of the chip using an optical assembly to measure the The first array of EWOD traps is then aligned to multiple microdroplets on the surface of the trap. forming a ray and using a second optical element to generate an oEWOD track on the surface of the chip. forming a second array of traps, wherein one or more of the OEWOD traps of the second array A step of aligning the plurality of oEWOD traps with a first array of oEWOD traps and Inspecting the contents of the liquid, and detecting whether one or more of the microdroplets are present in the second EWOD trap. adjusting the first optical assembly while held in place by the array; A method is provided which includes:

[0024] Both the first and second optical assemblies hold and manipulate microdroplets on the surface of the chip. By forming an array of oEWOD traps for During the stop or adjustment, one of the optical assemblies is used to locate all or selected portions of the microdroplets on the surface. This allows the microfluidic chip to be kept in a fixed position, greatly increasing its operational flexibility. This allows thousands of droplets to be dispensed without losing any droplets during interruptions required by adjustments to one side of the assembly. The droplets can be manipulated with different parameters or moved to different locations It is possible.

[0025] In some embodiments, the method comprises: Selecting a subset of microdroplets from the array and capturing the selected subset of microdroplets. deactivating all oEWOD traps except the one that captures the oEWOD trap; and and removing from the array of microdroplets those microdroplets that are not in the selected subset. This includes:

[0026] The OEWOD is used to sort non-selected droplets, such as those that are deemed undesirable during the sorting operation. A step of deactivating the wrap and a step of performing a flushing operation to remove unwanted microdroplets. Steps are feasible to carry out on a very large scale, such as in early screening assays. For example, the content of microdroplets can be examined to determine which microdroplets are empty and which are full. This may be a test to determine whether the target cells contain any of the target cells. The non-selected microdroplets to be sampled may be microdroplets that do not contain cells. The flushing operation removes droplets that are not in the subset and removes droplets that are in the subset. The first optical assembly's oEWOD trap is used to capture the droplets without being obstructed by the Sorting the array and / or deactivating the associated oEWOD traps Then, the continuous phase is introduced into the microfluidic chip via multiple fluid inlets and the selected subset is selected. Removing the microdroplets.

[0027] The initial step of the flushing operation is to sort the array and remove unwanted droplets from the selection sub- The droplets in the set are not disturbed, and unwanted microdroplets are further removed, especially during removal. The aim is to ensure that the droplets do not collide with the droplets marked for inspection. Sorting can be achieved by, for example, moving unwanted microdroplets in the center of the array to more unwanted microdroplets at the outer edge of the array. This may include replacing the selected microdroplets with the non-selected microdroplets for testing. The optical assembly is used to flush the selected droplets across the surface of the chip. No need to manipulate droplets and no need to deactivate each oEWOD trap to microdisplay a selected subset The need for fine-grained control of tiny droplets in large-scale operations, as only droplet position needs to be maintained The continuous phase is any one of silicone oil, mineral oil, and fluorocarbon oil. It may consist of:

[0028] In some embodiments, the adjustment of the first optical assembly includes changing the resolution, changing the magnification, Change the field of view, change the color selection element included in the assembly, and change the color closest to the sample being imaged. In some embodiments, the method includes at least one of replacing the lens assembly. The method further inspects the contents of the array of microdroplets using the first optical assembly after conditioning. The method further includes the step of:

[0029] As mentioned above, the method of the present invention is useful for microdroplet assays, particularly for large-scale microdroplet manipulation. In some embodiments, the method of the present invention includes: The assay parameters can be adjusted without losing droplets from the initial array that is formed. For example, a first optical assembly with a wide field of view can be used to capture thousands of microdroplets. First, an assay can be performed on an array containing droplets, and then a subset of those microdroplets can be used. They can be selected for further testing, allowing for subsequent more precise droplet manipulation or testing. The second optical assembly positions the selected microdroplet while reducing the field of view of the first optical assembly. This example is not limiting and may include microdroplets of an array of microdroplets. A selected subset or all of the optical elements are held in place by a second optical assembly while the subsequent optical elements are Applying the principle of adjusting the parameters of the first optical assembly between experiments, The method can increase the efficiency and fine-tune the microdroplet assay.

[0030] In some embodiments, the first optical assembly according to the present invention disclosed herein comprises , can be used to move, coalesce, and / or split microdroplets.

[0031] In some embodiments, the second optical assembly according to the present invention disclosed herein Microfluidic chips using optically mediated electrowetting (oEWOD) It can be used to manipulate microdroplets, such as moving, merging, and / or splitting microdroplets on a substrate. .

[0032] In some embodiments, the present invention provides a method for deactivating a first optical assembly and deactivating a second optical assembly. Using the oEWOD traps formed by assembly, The microfluidic chip further includes translating the array of microdroplets by a specific They often contain several different zones designed to perform different tasks. The surface of the droplet may be a classification zone, a test zone, or a zone suitable for assays using specific types of cells. The surface of the chip may include treated zones to allow for the operational flexibility of such assays. Another way that the efficiency can be enhanced by using a dual assembly configuration is to A selected subset of the array or microdroplets is then transferred to a second optical assembly either before or after performing the assay. The second optical assembly is used to transport the light between the zones. Translation of the entire array of traps does not require fine-grained, droplet-by-droplet control of oEWOD traps and the relative positions of the transported array droplets are maintained relative to each other. be.

[0033] In some embodiments, the first optical assembly provides more imaging power than the second optical assembly. High resolution. The dual assembly configuration of the present invention allows for the holding and transport of a large number of microdroplets at once. one optical assembly is designated as a generator of a "holding array" that can be transmitted; The other optical assembly is adapted to perform fine-grained control of the microdroplets in the array as required. It would be advantageous to involve a high-resolution adjustable array.

[0034] In some embodiments, forming the array of microdroplets comprises using a second optical assembly. The initial step is to form multiple oEWOD traps in the shape of the target array using an assembly. A first optical assembly is used to determine the location of a plurality of microdroplets on the surface of the microfluidic chip. The initial step of determining the number of oEWOD traps formed by the first assembly is used. By using the oEWOD, multiple microdroplets are manipulated into an array that matches the target array of the oEWOD trap. This operation requires precise droplet control, but the chip configuration allows for This is a reliable method for forming an array.

[0035] In another embodiment, the step of forming the array of microdroplets comprises: forming a first array of oEWOD traps using and dispensing a plurality of microdroplets onto the surface of the chip on which the first array is located. The microdroplets positioned on the surface of the chip are collected at the oEWOD trap array position. Forming an array of oEWOD traps on the surface of the chip allows for the Eliminates the need for precise droplet-by-droplet control, allowing for micro-analysis in assays with many micro-droplets of analysis. This is an efficient method for forming arrays of small droplets. The pores can be formed quickly and easily.

[0036] In some embodiments, the electromagnetic radiation from the first optical assembly is directed to the microdroplets and multiplexed with electromagnetic radiation from an inspection component configured to inspect the contents thereof. Combining the optical assembly that forms the EWOD trap with the inspection component This allows for more efficient direction of the inspection radiation.

[0037] In some embodiments, the examination of the contents of the microdroplets can be performed using techniques such as fluorescent imaging, metal nanoimaging, or the like. Localized optical plasmon resonance in nanoparticles, FRET, dark field, bright field, Raman, absorption, quantum dots The method is carried out using at least one of the following techniques: fluorescence, spectroscopy, and / or fluorometric methods. Fluorescence-based methods are particularly advantageous. If the investigation metric is the localized optical plasmon resonance at the metal nanoparticle, the particle The surface is functionalized with a target molecule or antibody, and the detection method is based on the functionalized nanoparticles in response to the binding of the target molecule to the surface. Detects changes in the spectral response of nanoparticles.

[0038] In some embodiments, at least one of the first and second arrays of oEWOD traps On the one hand, spatial light modulating devices such as TFT, DMD projectors, DLV, and LCoS projectors LEDs, OLEDs, CRTs, projectors with screens, and micro LED arrays The projection optical system is formed by using at least one of a light emitting array and a projection optical system.

[0039] According to another aspect of the present invention, there is provided an apparatus for manipulating microdroplets, the apparatus comprising: a first and second composite wall defining a first surface and a second surface, the first and second composite walls defining a first surface and a second surface, the second surface and the second surface being optically mediated electrowetting (o configured to manipulate microdroplets on a surface defining a microfluidic space by EWOD. In addition, a microfluidic chip and a plurality of microscopic particles on a surface are formed by forming a plurality of first EWOD traps. a first optical assembly configured to manipulate small droplets; and a second optical assembly configured to manipulate a plurality of second optical droplets on a surface. forming a wrap to separate a plurality of microdroplets during the adjustment and / or injection operation of the first optical assembly; a second optical assembly configured to maintain a relative position and a second optical assembly configured to adjust the content of the plurality of microdroplets; and an inspection component configured to inspect the

[0040] In some embodiments, the inspection component is an electromagnetic radiation source and the first optical assembly The electromagnetic radiation from the assembly is multiplexed.

[0041] In some embodiments, the first and second composite walls are at least partially transparent; The first and second optical assemblies are positioned on opposite sides of the microfluidic space. At least one of the first and second optical composite walls is transparent, and the first and second optical composite walls are The second optical assembly is positioned on the same side of the microfluidic space, and the color filters are positioned on the second optical assembly. to prevent interference with the first optical assembly.

[0042] In some embodiments, at least one of the first and second optical assemblies includes a microphone. In some embodiments, the second optical assembly or both optical assemblies include a lens array. The optical assembly provides relatively coarse-grained optical control and does not necessarily control all the illumination spots independently. Rather than being switched, they are arranged in small banks that can be activated separately.

[0043] In some embodiments, the device comprises an external flow control valve and pump set and a dosing and an arrangement of inlets for inlet and flushing operations.

[0044] In some embodiments, the optical assembly has a diameter of between 20 μm and 250 μm. The device is configured to provide an array of spots with a pitch of 50 μm to 675 μm, particularly 30 The diameter is 30 μm to 250 μm and the pitch is 30 μm to 300 μm. Typically, the diameter is 2.5 times the droplet diameter. In some embodiments, the optical assembly A spot array with an approximate pitch of 120 μm or 1 μm and a spot size of about 50 μm. In some embodiments, the optical assembly is configured to provide a 5 μm configured to provide spot arrays with diameters of ∼30 μm, and 12.5 μm to 75 μm They are formed at a pitch of 1 μm.

[0045] In some embodiments, the methods of the present invention involve dielectrophoresis of microparticles in droplets.

[0023] The present invention is applicable to photoactive devices, such as devices configured to manipulate microparticles comprising The cells or particles are then subjected to a functionally identical optical device for generating a virtual dielectrophoretic gradient. Microparticles, as defined herein, are used to manipulate and examine biological cells, polystyrene, and other materials. Microbeads made of materials including polyethylene and latex, magnetic microbeads, or colloids It may refer to particles such as cellulose.

[0046] Similar to the method described above for photoelectrowetting, a high-resolution first optical assembly using a combination of optically mediated dielectrophoresis to move particles and / or cells. Fine manipulation and detailed inspection are performed. A coarse second optical assembly is used to visualize the dielectrophoretic transistor. By combining these two assemblies, the method The optical assembly is used for scanning and inspection operations while the coarse optical assembly is used for non-scanning. It is possible to hold and transport a large number of particles and / or cells at any one time.

[0047] Therefore, according to another aspect of the present invention, there is provided an apparatus for manipulating microparticles, comprising: a first and second composite transparent wall defining a space and positioned on a surface defining the retention space; a tip configured to manipulate the microparticles and to direct light to the surface through the first composite wall; Directing the beam to form multiple primary optical traps to manipulate multiple microparticles on a surface a first optical assembly configured to direct a light beam to the surface through the second composite wall; forming a plurality of second optical traps on the surface during adjustment and / or introduction of the first optical assembly; a second optical assembly configured to maintain a relative position of the plurality of microparticles during operation; and an inspection component configured to examine the contents of the plurality of microparticles; An apparatus is provided. [Brief explanation of the drawings]

[0048] [Figure 1] 1 shows a cross-sectional view of an exemplary microfluidic chip described in Patent Document 1. [Figure 2] 1 shows a cross-sectional view of an exemplary microfluidic chip suitable for carrying out the methods of the present invention. [Figure 3] A top view of the surface of a microfluidic space is shown. [Figure 4A] 10 shows an example of a process for a drop action and droplet investigation workflow. [Figure 4B] 10 shows an example of a process for a drop action and droplet investigation workflow. [Figure 4C] 10 shows an example of a process for a drop action and droplet investigation workflow. [Figure 4D] 10 shows an example of a process for a drop action and droplet investigation workflow. [Figure 4E] 10 shows an example of a process for a drop action and droplet investigation workflow. [Figure 5A] 1 shows a diagram of droplet coalescence according to the present invention; [Figure 5B] 1 shows a diagram of droplet coalescence according to the present invention; [Figure 5C] 1 shows a diagram of a droplet splitting operation according to the present invention; [Figure 5D] 1 shows a diagram of a droplet splitting operation according to the present invention; [Figure 6A] 10A-10C show alternative views of droplet coalescence in accordance with the present invention; [Figure 6B] 10A-10C show alternative views of droplet coalescence in accordance with the present invention; [Figure 6C] 10 shows an alternative view of a droplet splitting operation in accordance with the present invention. [Figure 6D]10 shows an alternative view of a droplet splitting operation in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0049] To further illustrate various exemplary aspects of the present disclosure, specific embodiments of the present disclosure will now be described. will be described in detail with the accompanying drawings.

[0050] Referring to FIG. 1, an oEW suitable for high-speed manipulation of water microdroplets, as described in Patent Document 1, is shown. A cross-sectional view of an exemplary microfluidic chip device with OD structures is shown.

[0051] The device is covered with a transparent layer 15 of conductive indium tin oxide (ITO) with a thickness of 130 nm. The glass substrate is provided with an upper glass plate 13 and a lower glass plate 14 each having a thickness of 500 μm. Each is connected to an A / C source 16 with the ITO layer on the bottom glass plate 14 as the ground. The upper glass plate 13 is coated with an 800 nm thick amorphous silicon layer 17. The amorphous silicon layer 17 is made of high-purity alumina or hafnium, each having a thickness of 160 nm. The alumina / hafnia layer 18 further renders the surface of the alumina / hafnia layer 18 hydrophobic. Supporting trichloro(1H,1H,2H,2H-perfluorooctyl)silane layer 19 The upper glass plate 13 and the amorphous silicon layer 17 are coated with an intervening silicon dioxide layer. A spacer was used to ensure that the droplets were compressed to some extent during their introduction into the device. m apart.

[0052] A first optical assembly, in this example a reflective pixelated screen illuminated by an LED light source 20 The image of the lens is positioned approximately below the lower glass plate 14 and illuminated at 0.01 Wcm -2 Level of visibility Light (wavelength 660 nm or 830 nm) is emitted from each diode 21 and penetrates the lower layer 14 and The incident light beam propagates through 15 in the direction of the arrows. At various points of incidence, photoexcited charge regions 22 are generated in the amorphous silicon layer 17, which then This induces a change in the solid-liquid contact angle at the corresponding electrowetting position 23 of the Na / hafnia layer 18. The oEWOD traps are then formed at these positions. 23 to hold the microdroplet 2 in place or to move the microdroplet 2 from one point 23 to another. The optical assembly 20 provides the necessary capillary force to propel the diodes 21 of the array. A pre-programmed algorithm determines which of the following will be irradiated at any given time. It is controlled by a microprocessor 24.

[0053] Referring to FIG. 2, a microfluidic chip having the same or similar stack structure as the chip of FIG. A cross-section of the chip is shown, showing a first optical assembly 20 as well as a number of microdroplets 2. A separately controllable second optical assembly for increased flexibility and capabilities in operations including processing. The Umbri 25 has been introduced.

[0054] The light source from the second optical assembly is activated and held by the first light source during the throw-in option. and / or the first optical assembly. During adjustment of the first light source, for example, during switching of the lens of the first light source, during investigation, or during the surface of the microfluidic space The microdroplet array can act as a supporting light source, such as during translation of the microdroplet array through a

[0055] The light source of the optical assembly does not necessarily have to be an LED light source. Any optical arrangement that can be used to project a programmable light spot is suitable. For example, the projection optics may be combined with a microlens array arrangement or a fly's eye arrangement. In the example of the present disclosure, the optical electro-weave The lighting pattern is generated by, for example, a digital micromirror device, an LCD display, Using a spatial light modulator or LED array, the object plane, i.e., the EWOD trap The projected spots are spatially modulated across the plane of the formed surface. An exemplary array of projected spots has a diameter of It may consist of spots with a 50 μm pitch, i.e., a center-to-center distance between spots of 100 μm. do.

[0056] In some examples, the device of FIG. 2 may be configured to emit light suitable for photo-electrowetting operation. Equipped with a combined inspection and manipulation optical assembly, multiplexed with light suitable for fluorescence excitation. , for example, when the investigated component is a passive light collecting system rather than an electromagnetic radiation source, The components may be physically separated.

[0057] Referring to FIG. 3, a top view of the surface of a microfluidic space into which a large number of microdroplets have been introduced is shown. In this example, the field of view of the first optical assembly 20, i.e., the microdroplet 2 on the surface The affected area is indicated by a first boundary 26, and the field of view of the second optical assembly 25 is indicated by a second boundary 27. In the exemplary configuration of FIG. 3, the first optical assembly has a much narrower field of view, Therefore, it is more suitable for the fine manipulation of a small number of microdroplets, whereas the second optical assembly is a much broader technique suitable for maintaining many microdroplets in place over a large portion of a surface. It can be seen that it has a wide field of view.

[0058] Typically, inspection components are designed to maximize light collection efficiency and fluorescence imaging resolution. By switching between these objective lenses, the imaging Resolution and concentration during assays to examine the contents of microdroplets on the chip with increasing and decreasing magnification. It is possible to increase or decrease the light efficiency. High magnification inevitably reduces the field of view of the imaging system. It could be.

[0059] When the inspection optical path and the optical electrowetting manipulation optical path are multiplexed, the field of view is reduced. As a result, the droplets that were held in place by the operation pattern are outside the field of view of the optical system, and this They can move between them by diffusion or fluid flow.

[0060] Similarly, the process of changing objective lenses temporarily interrupts the operating pattern, In addition, in some cases, the light from the scanning pattern may be dissipated. Discontinue the optical manipulation light during fluorescence imaging to prevent it from interfering with the fluorescence image. It may be necessary to perform this procedure, but the droplet may move uncontrolled during this long pause. do.

[0061] A low-resolution spot-generating optical assembly is combined with a high-resolution inspection and manipulation assembly. By using a holographic technique, it is possible to overcome these limitations.

[0062] In an exemplary process, droplets are captured in a microfluidic chamber using a high-resolution optical assembly. The image is then captured after being positioned in a specific layout by the camera. The pattern is generated by a low-resolution spot generating assembly. This pattern is activated as a holding pattern when changing the objective lens of the inspection assembly. It can also be used to hold droplets that fall outside the field of view during fluorescence acquisition. Alternatively, in some embodiments, the droplet may be held in a low High-resolution optical assembly positioned in a specific layout by the high-resolution optical assembly It can be inspected by

[0063] The process involves software generating pixel maps on the target surface from both illumination sources. Controlled by performing a 2D coordinate transformation between two light sources and then applying this to a high-resolution source Coordinate transformations can be performed using pixel scaling and translation between two projectors, as well as high-resolution Consider the range of resolution light sources and various objective lenses.

[0064] If a broadband light source is selected for the low-resolution optical assembly, certain bands of the spectrum will be filtered out. blocking "notch" that allows light outside the band to be used for the retention pattern Applying a filter to the input light prevents interference with the light used for fluorescence imaging. It is possible to burn it.

[0065] Additionally, a low-resolution optical assembly is used to maintain the relative position between droplets during sample movement. For example, a mechanical motorized motion stage can be used to translate the sample. If it is necessary to move the droplets, the droplets are gradually moved to the stage so that the relative position of the droplets remains unchanged. It is possible to shift the pattern registration so that they move in unison.

[0066] Two photoelectrowetting control panels (from high-resolution and low-resolution assemblies) To overlap the turns, a substantially transparent optical electrowetting device is used. It is preferable to have one pattern projected from each side of the device. As a result, the low-resolution pattern is from the same side as the high-resolution pattern, but This can be applied at an oblique angle so that the light falls outside the numerical aperture of the objective. In this case, adaptive optics can be added to a low-resolution projector to reduce the image distortion caused by oblique projection angles. It is preferable to adjust the shape and focus of the projected pattern to avoid image distortion.

[0067] 4A-4E, it is possible to achieve, for example, over a million droplets without the need for precise droplet-by-droplet control. Another exemplary drop operation and droplet investigation workflow suitable for processing a large number of microdroplets is shown. The process is shown.

[0068] Referring to FIG. 4A, in a first step, the optical assembly projects light in the form of an array. The shadowed light spot causes photoexcitation of the photoactive layer, resulting in the deposition of charge on the surface of the dielectric layer. This is due to the accumulation of microdroplets on the surface of the microfluidic space in the microfluidic chip. It acts as an optical trap (oEWOD trap).

[0069] Referring to FIG. 4B, in a second step, dropletized biological agents, such as cells, are is a continuous phase flow that may 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 The device deposits microdroplets in a pattern that matches the array pattern of the projection optics (optical assembly). Arrange yourself and organize.

[0070] Referring to FIG. 4C, the array of trapped microdroplets is then transferred to the permeable membrane of the microfluidic chip. The droplets are probed through a transparent substrate, and the results of the probe are used to select a subset of droplets based on a predetermined metric. This metric is useful for, for example, fluorescent imaging or display with metal nanoparticles. It may be a surface plasmon resonance.

[0071] Referring to FIG. 4D, once a subset of microdroplets has been selected, any missing droplets not in the selected subset are removed. The light spot corresponding to the desired microdroplet is turned off and pumped to its destination via a series of pumps and valves. A continuous flow is introduced into the microfluidic chip at 1000 kJ / s, and the corresponding microdroplets are deposited on the interrogation zones on the surface of the chip. The cells are then removed from the cell and, in some cases, completely removed from the microfluidic chip.

[0072] Referring to Figure 4E, the set of selected microdroplets is subsequently collected for further post-processing or analysis. The droplets are then either flowed out of the chip or transported by a light spot using a micromirror array. The cells are then kept on the chip for further processing and analysis using the aforementioned arrangements that control their movement. .

[0073] In addition to the embodiment shown in FIG. 1, other oEWOD microfluidic chips suitable for carrying out the methods of the present invention are also available. The general characteristics and optional features of the group and the composite structures contained therein are described below.

[0074] The EWOD structure comprises a first substrate, a transparent insulating film having a thickness ranging from 70 nm to 250 nm on the substrate, and A clear first conductive layer, activated by electromagnetic radiation in the wavelength range of 400nm to 850nm on the conductive layer a photoactive layer having a thickness in the range of 300 nm to 1500 nm and a 30 a first composite wall comprising a first dielectric layer having a thickness in the range of 100 nm to 160 nm; and a second substrate; a second conductor layer having a thickness in the range of 70 nm to 250 nm on the substrate; and a second composite wall consisting of an optional second dielectric layer having a thickness in the range of 0 nm to 160 nm; The exposed surfaces of the first and second dielectric layers are spaced apart by 20 μm to 180 μm. and defining a microfluidic space adapted to contain the first and second composite walls. The A / C source supplies the voltage connecting the two conductor layers, and the corresponding virtual electrons are incident on the photoactive layer. a photoactive layer adapted to induce a trowetting position on the surface of the first dielectric layer; a first and second electromagnetic radiation source having an energy greater than the band gap; At least one method for moving microdroplets by changing the location of the microwetting location. The point of incidence of electromagnetic radiation on the photoactive layer is adjusted to create one electrowetting path. The first and second walls of these structures sandwich a microfluidic space therebetween. And it is transparent.

[0075] The first and second substrates are made of a material with high mechanical strength, such as glass, metal, or engineering Suitably, the substrate is made of plastic. In yet another embodiment, the first and second substrates may have a flexibility of 100 μm. In some embodiments, the first substrate has a thickness in the range of 1000 μm to 1000 μm. In some embodiments, the second substrate is made of one of a quartz glass, a fused silica, and a glass. is made of quartz glass or glass.

[0076] The first and second conductor layers are positioned on one side of the first and second substrates, and are typically 70 nm to 2 At least these layers have a thickness of 50 nm, preferably in the range of 70 nm to 150 nm. On the other hand, transparent conductive materials such as indium tin oxide (ITO) and very thin conductive materials such as silver are used. They are made of metal films or conductive polymers such as PEDOT. These layers can be continuous sheets or Alternatively, the conductor layer may be formed as a network of discrete structures such as wires. It may be a mesh of conductive material with openings.

[0077] The photoactive layer generates localized regions of change in response to stimulation by a second electromagnetic radiation source. For example, a semiconductor material with a wavelength of 300 nm to 1500 nm is suitable. In some embodiments, the hydrogenated amorphous silicon layer may have a thickness in the range of 0.1 to 1.0 μm. In the first wall case, the photoactive layer is activated by the use of visible light. In some cases, the conductor layer in the case of the second wall is a dielectric layer, typically with a thickness in the range of 30 nm to 160 nm. The dielectric properties of this layer are 7 High dielectric strength exceeding 3 V / m Preferably, this is as consistent as possible with avoiding dielectric breakdown. In some embodiments, the dielectric layer is made of alumina, silica, hafnia, or the like. or a non-conductive polymer thin film.

[0078] In another embodiment of these structures, at least the first dielectric layer, and preferably both dielectrics The conductive layer is coated with an antifouling layer to allow desired microelectrode formation at various virtual electrowetting electrode positions. It helps establish the droplet / carrier fluid / surface contact angle and also allows the droplet to transfer to the chip. The second wall prevents the contents of the microdroplets from being attached to the surface and reduced when the microdroplets are moved. If no layer is included, the second antifouling layer may be applied directly to the second conductor layer.

[0079] For optimal performance, the antifouling layer should be measured at 250°C as a three-point air-liquid-surface interface. In this case, the droplet / carrier fluid / surface contact angle should be in the range of 50 to 180°. In some embodiments, these layer(s) may be In another embodiment, these layers have a thickness of less than 100 nm and are typically monolayers. , hydrophilic groups, such as methyl methacrylate or its derivatives substituted with alkoxysilyl groups, Consists of a polymer of acrylic ester. Either or both of the anti-fouling layers ensure optimal performance. In some embodiments, the cross-linking is chemically compatible. To achieve this, an intervening layer of silica less than 20 nm thick is sandwiched between the antifouling coating and the dielectric layer. obtain.

[0080] The first and second dielectric layers, and therefore the first and second walls, have a width of 10 μm or more, preferably It defines a microfluidic space that accommodates microdroplets ranging from 20 μm to 180 μm. Alternatively, the droplet itself may be larger than 10% of the droplet space width before being accommodated. Suitably, the droplets have a characteristic diameter that is greater than 20%. The compression of the electrowetting In some embodiments, the first and second dielectric layers are fluororesin. It is coated with a hydrophobic coating such as silane.

[0081] In another embodiment, the microfluidic space maintains the first and second walls a predetermined amount apart. The spacer may include one or more spacers for supporting the light. Examples of the bead, pillar, and ridge formed from the intermediate resist layer produced by Alternatively, the spacers may be fabricated using deposited materials such as silicon dioxide or silicon nitride. Alternatively, a flexible plastic film with or without an adhesive coating may be used. The spacer layer can be formed using a film layer containing a variety of spacer geometries. This allows for the creation of narrow, tapered, or partially enclosed channels defined by rows of pillars. With careful design, these spacers can be used to control the flow of microdroplets. It is possible to aid in the formation of microdroplets, followed by microdroplet splitting and manipulation of the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of the chip, providing spacing between droplet populations. This prevents cross-contamination and directs droplet flow in the correct direction when inserting the tip under liquid pressure. can.

[0082] The first and second walls provide a potential difference between them, suitably in the range of 10 volts to 50 volts. These are biased using an A / C power supply attached to the conductor layer. The EWOD structure has a wavelength and optical activity in the range of 400 nm to 850 nm, preferably 660 nm. It is usually used in conjunction with a second electromagnetic radiation source having an energy above the band gap of the conductive layer. The photoactive layer is used when the incident intensity of the radiation is 0.01 Wcm -2 ~0.2Wc m -2 It is suitable to activate the virtual electrowetting electrode position in the range do.

[0083] If the electromagnetic radiation source is pixelated, the digital image will be illuminated by light from an LED or other lamp. Direct or indirect use of a reflective screen such as a digital micromirror device (DMD) This allows for very accurate positioning of the virtual electrowetting electrodes. Complex patterns can be rapidly formed and destroyed on the first dielectric layer, thereby Tightly controlled electrowetting forces can be used to saturate microdroplets into essentially any virtual Such electrowetting paths can be precisely steered along the path. It is assumed that the first dielectric layer consists of a series of virtual electrowetting electrode locations. You can see it here.

[0084] The point of incidence of the electromagnetic radiation source on the photoactive layer may be of any convenient shape, including conventional circular or annular shapes. In some embodiments, the shape of these points can be determined by the corresponding pixel. In another embodiment, the droplets are placed in a microfluidic space. In one embodiment, the point of incidence and therefore the electron The trowetting electrode position may be crescent-shaped and point in the desired direction of travel of the microdroplets. The electrowetting electrode position itself is smaller than the surface of the microdroplet adhering to the first wall. and provide a maximum electric field strength gradient across the contact line formed between the droplet and the surface dielectric. It is appropriate to do so.

[0085] In some embodiments of the oEWOD structure, the second wall may be connected to the same or a different electromagnetic radiation source. This allows the virtual electrowetting electrode position to be guided even in the second dielectric layer. The addition of a second dielectric layer also improves the wetting perimeter of the microdroplets from the top to the bottom of the structure. This allows for the transfer of the liquid to the surface and the application of a larger electrowetting force to each droplet. .

[0086] The first and second dielectric layers may consist of a single dielectric material, or may consist of two or more dielectric materials. The dielectric layer may be a composite of materials such as, but not limited to, Al2O3 and SiO2. It's okay to come.

[0087] A structure may be provided between the first and second dielectric layers. It is not limited to, but may be made of epoxy, polymer, silicone or glass, or a mixture thereof. or composite materials and may have straight, sloped, curved, or microstructured walls / surfaces. The structure between the first and second dielectric layers is connected to the upper and lower composite walls to form a sealed microphone. A bi-fluidic device is created to define channels and regions within the device. This structure is Alternatively or additionally, the conductor and dielectric may occupy the gap between the walls. The material may be deposited on a shaped substrate.

[0088] Some aspects of the method and apparatus of the present invention may be used with devices other than electrowetting devices. Optically active devices such as devices configured to manipulate microdroplets by dielectrophoresis or optical tweezers In such devices, cells or particles are induced by virtual light. It is operated and inspected using functionally identical optical equipment for generating electrophoretic gradients. Microparticles, as defined herein, are made from materials including biological cells, polystyrene, and latex. This refers to particles such as microbeads, hydrogels, magnetic microbeads, or colloids made of Dielectrophoresis and optical tweezers mechanisms are known in the art and can be implemented by those skilled in the art. It can be easily implemented.

[0089] Similar to the invention described above for photoelectrowetting, a first optical axis with high resolution is Assembly and the combination of optically mediated dielectrophoresis to separate particles and / or cells A second optical assembly is used to perform detailed manipulation and inspection of the dielectrophoretic tracer. By combining these two assemblies, the method: The coarse optical assembly is used while the fine optical assembly is used for manipulation and inspection operations. They are capable of holding and transporting very large numbers of particles and / or cells.

[0090] Referring to FIGS. 5A and 5B, the droplet is then passed through a second optical setup as described herein. and a first optical setup as described herein. 5A shows a diagram of the merging operation using a The arrows in Figure 5A show the droplets before coalescence. Figure 5B shows the droplets after coalescence. 1 shows the coalesced droplets after operation.

[0091] Referring to FIGS. 5C and 5D, the droplets are then passed through a second optical setup as described herein. and a first optical setup as described herein. 5C shows a diagram of the splitting operation using the .times. ... The arrows indicate the direction of droplet splitting to provide additional droplets. Shows post-split events.

[0092] Referring to Figures 6A and 6B, the droplet is held between movements by a second optical setup 52. and combined in operation using a second optical setup 52 as described herein. 6A, a diagram of the coalescence operation is shown. The arrows in FIG. 6A indicate the direction of the droplets during the coalescence operation. FIG. 6B shows the coalesced droplets after the coalescence operation.

[0093] 6C and 6D, the second optical setup 52 holds the image between operations. A diagram of the droplet splitting operation is shown, where the droplet is split using the second optical setup. The arrows in FIG. 6C indicate droplet division during the splitting operation to form additional droplets. The splitting direction is shown in Figure 6D. Figure 6D shows multiple droplets formed after the splitting operation.

[0094] The optical assembly that inspects a subset of the array is made up of an optical assembly that holds the array in place. It has a much smaller field of view than Swertia japonica. Within the reduced field of view, only one microdroplet is visible. Alternatively, the inspection optical assembly may have 24, 48, or 2 There may be 56, 1048, or any suitable number of microdroplets. The process is performed one at a time, with the optical assembly scanning the field of view to inspect each microdroplet sequentially. With respect to the optical system, the optical system is in a single location and the scanning is performed by a camera that forms part of the optical assembly. Part of the FOV by processing information from a part of the image projected onto the image sensor, such as Alternatively or additionally, the optical assembly may be configured to integrate its entire field of view. This coarse-grained data is the most informative of the array. It can be combined with a microdroplet-level review to quickly focus on key areas. Cut.

[0095] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. cormorant.

[0096] As used herein, "and / or" refers to two specified features either with or without the other. or a specific disclosure of each of the components. For example, "A and "and / or B" means (i) A, (i i) B, and (iii) A and B shall be deemed to be specific disclosures of each of them.

[0097] Unless the context dictates otherwise, the above feature descriptions and definitions do not apply to any particular embodiment of the invention. The present invention is not limited to any aspect or embodiment, but applies equally to all aspects and embodiments described. .

[0098] As will be further understood by those skilled in the art, the present invention will now be described by way of example with reference to several embodiments. Although described, it is understood that the present invention is not limited to the disclosed embodiments, but extends to the scope of the invention as defined by the appended claims. Alternate embodiments may be constructed without departing from the scope.

Claims

1. Optically mediated electrowetting (oEWOD) for microfluidic chips 1. A method for inspecting and / or selecting microdroplets on a substrate, comprising: A plurality of oEWOD traps are temporarily formed on the surface of the chip, and causing the plurality of microdroplets on the surface to form an array of microdroplets; Preserving the entire array of microdroplets while inspecting at least a subset of the array. Steps to maintain A method comprising:

2. 10. The method of claim 1, wherein the step of holding the entire array of microdroplets comprises: a substep of temporarily forming a second array of oEWOD traps on the surface of the chip; Pu and, one or more of the oEWO traps of the second array connected to the oEWO traps of the first array; a substep of aligning with the D trap; How to make it easier.

3. 3. The method of claim 1, wherein the plurality of oEWOD traps are temporarily formed. the step of performing the step of detecting a second array of oEWOD traps is performed by an optical assembly. wherein the step of temporarily forming is performed by a second optical assembly.

4. 4. The method of claim 3 when dependent on claim 2, wherein the oE of the second array Aligning one or more of the WOD traps with the oEWOD traps of the first array. The step of displacing the microdroplets between the first optical assembly and the second optical assembly includes: A method that allows for the step of handing off retention of an entire array.

5. The method according to any one of claims 1 to 4, One or more of the microdroplets are held in place by a second array of oEWOD traps. adjusting the first optical assembly while The method further comprises:

6. The method according to any one of claims 1 to 5, selecting a subset of microdroplets from the array of microdroplets based on an examination of the contents of the microdroplets; selecting a Deactivating all oEWOD traps except those capturing a select subset of microdroplets and A flushing operation is performed to remove the microdroplets not in the selected subset from the array of microdroplets. Step of removing from The method further comprises manipulating said microdroplets with

7. 7. The method of claim 6 when dependent on claim 3, wherein the flushing operation comprises: The removal of the microdroplets not in the subset is performed by the microdroplets in the subset. The oEWOD trap of the first optical assembly or the second assembly is arranged to be unobstructed. and / or Once the associated oEWOD trap is deactivated, the microphone is (b) introducing a continuous phase into the fluidic chip to remove microdroplets not in said selected subset; A method comprising:

8. 6. The method of claim 5, wherein the adjustment of the first optical assembly includes changing the resolution, doubling the resolution, or adjusting the optical axis. changing the ratio, changing the field of view, changing the illumination source, changing the color selection element included in the assembly, and and replacing the lens assembly closest to the sample being imaged.

9. 6. The method of claim 5, wherein after said adjustment, said first optical assembly is used to The method further comprising the step of performing a further inspection of the contents of the array of droplets.

10. 4. The method of claim 3, wherein the first optical assembly is stopped and the second optical assembly is stopped. The oEWOD trap formed by the bridge is used to attach the trap to the surface of the microfluidic chip. translating the array of microdroplets across a

11. 4. The method of claim 3, wherein the step of forming an array of microdroplets comprises: forming a plurality of oEWOD traps in the shape of the target array using the second optical assembly; an initial step of a first optical assembly to detect the plurality of microdroplets on the surface of the microfluidic chip; The initial step of determining the location; The plurality of oEWOD traps formed by the first assembly are used to Initial manipulation of droplets into an array that matches the target array of oEWOD traps. Steps and A method comprising:

12. 4. The method of claim 3, wherein the step of forming an array of microdroplets comprises: forming a first array of oEWOD traps using a first optical assembly; The plurality of oEWOD traps are positioned on the surface of the chip where the first array of oEWOD traps is located. and injecting the microdroplets of A method comprising:

13. The method according to any one of claims 1 to 12, wherein the detection of the contents of the microdroplets is The investigations were carried out using fluorescence imaging, localized optical plasmon resonance on metal nanoparticles, FRET, and dark field. The method is performed using at least one of bright field, Raman, absorption, and quantum dot fluorescence spectroscopy. method.

14. An apparatus for manipulating microdroplets, comprising: a first and second composite wall defining a microfluidic space and configured to receive an optically mediated electrical current; microfluidic space on the surface that defines the microfluidic space by trowetting (oEWOD) a microfluidic chip configured to manipulate droplets; forming a plurality of first EWOD traps to manipulate a plurality of microdroplets on the surface; a first optical assembly configured; forming a plurality of second oEWOD traps on the surface during adjustment of the first optical assembly; and / or a second light source configured to maintain the relative positions of the plurality of microdroplets during the dispensing operation. academic assembly, an inspection component configured to examine the contents of the plurality of microdroplets; A device comprising:

15. 15. The apparatus of claim 14, wherein the inspection component is an electromagnetic radiation source, and the The device is multiplexed with electromagnetic radiation from the first optical assembly.

16. 16. The device of claim 14 or 15, wherein the first and second composite walls are at least partially the first and second optical assemblies are positioned on either side of the microfluidic space. Attachable device.

17. 16. The device according to claim 14 or 15, wherein at least one of the first and second optically composite walls is transparent, and the first and second optical assemblies are located on the same side of the microfluidic space. and a color filter is applied to the second optical assembly to A device that prevents interference.

18. 18. The apparatus according to any one of claims 14 to 17, wherein the first and second optical assemblies At least one of the lenses includes a microlens array.

19. 1. An apparatus for manipulating microparticles, comprising: a surface defining said retention space, said surface including first and second transparent composite walls defining said retention space; a chip configured to manipulate microparticles positioned thereon; Directing a light beam through the first composite wall onto the surface to form a plurality of first light traps. a first optical assembly configured to manipulate a plurality of microparticles on the surface; and Directing a light beam through the second composite wall onto the surface to form a plurality of second light traces on the surface. forming a gap between the first optical assembly and the plurality of microphones during adjustment and / or insertion of the first optical assembly; a second optical assembly configured to maintain the relative positions of the black particles; an inspection component configured to examine the contents of the plurality of microparticles; A device comprising:

20. The method according to any one of claims 14 to 19, wherein the first or second optical assemblies The projector is a spatial light modulating device such as a TFT, DMD projector, DLV, and LCoS projector. LEDs, OLEDs, CRTs, projectors with screens, and micro LED arrays The method includes a projection optical system including at least one light emitting array.

Images