Devices and methods for detecting microdroplets of cells

Abstract

To provide devices and related methods for the rapid identification, manipulation, and selection of cells. [Solution] A device, system, and associated method are provided for manipulating and / or determining one or more properties of cells contained in a biological sample. In particular, a device and a method of using the same are provided, the device including: a sorting component configured to separate cell-containing microdroplets from empty ones into a population of first cell-containing microdroplets; a microdroplet manipulation component configured to manipulate the first microdroplets using real or virtual electrode wetting electrodes; and an optical detection system configured to detect optical signals from microdroplets through one or more detection windows.

Description

Technical Field

[0001] According to the present invention, devices for the rapid identification, manipulation, and selection of cells are provided. It is particularly useful for the manipulation of mammalian cells, either from immortalized cell culture samples or directly from tissue samples. It is also particularly useful for the rapid and parallel screening of patient specimens that are thought to contain evidence of infection. and related methods. It is particularly useful for the manipulation of mammalian cells, either from immortalized cell culture samples or directly from tissue samples. It is also particularly useful for the rapid and parallel screening of patient specimens that are thought to contain evidence of infection. and related methods. It is particularly useful for the manipulation of mammalian cells, either from immortalized cell culture samples or directly from tissue samples. It is also particularly useful for the rapid and parallel screening of patient specimens that are thought to contain evidence of infection. and related methods. It is particularly useful for the manipulation of mammalian cells, either from immortalized cell culture samples or directly from tissue samples. It is also particularly useful for the rapid and parallel screening of patient specimens that are thought to contain evidence of infection. and related methods. It is particularly useful for the manipulation of mammalian cells, either from immortalized cell culture samples or directly from tissue samples. It is also particularly useful for the rapid and parallel screening of patient specimens that are thought to contain evidence of infection.

Background Art

[0002] Devices for manipulating droplets or magnetic beads have been previously described in the art; see, for example, US6565727, US20130233425, and US20150 027889. In the case of droplets, this result can typically be achieved, for example, by moving the droplets through a microfluidic space defined by two opposing walls of a cartridge or a microfluidic tube, in the presence of immiscible carrier fluids. One or both of these walls are embedded with microelectrodes, which are covered with a dielectric layer, and each dielectric layer can be rapidly turned on / off at intervals to modify the electric field characteristics of the layer, and is connected to an A / C bias circuit. This creates a localized directional capillary force in the vicinity of the microelectrodes, which can be used to steer the droplets along one or more predetermined paths. Devices such as those using what is hereinafter referred to as "real" electrowetting electrodes in connection with the present invention are known in the art as abbreviated EWOD (Electrowetting on Dielectric) devices. This creates a localized directional capillary force in the vicinity of the microelectrodes, which can be used to steer the droplets along one or more predetermined paths. Devices such as those using what is hereinafter referred to as "real" electrowetting electrodes in connection with the present invention are known in the art as abbreviated EWOD (Electrowetting on Dielectric) devices. This creates a localized directional capillary force in the vicinity of the microelectrodes, which can be used to steer the droplets along one or more predetermined paths. This creates a localized directional capillary force in the vicinity of the microelectrodes, which can be used to steer the droplets along one or more predetermined paths. Devices such as those using what is hereinafter referred to as "real" electrowetting electrodes in connection with the present invention are known in the art as abbreviated EWOD (Electrowetting on Dielectric) devices. Devices such as those using what is hereinafter referred to as "real" electrowetting electrodes in connection with the present invention are known in the art as abbreviated EWOD (Electrowetting on Dielectric) devices. Devices such as those using what is hereinafter referred to as "real" electrowetting electrodes in connection with the present invention are known in the art as abbreviated EWOD (Electrowetting on Dielectric) devices.

[0003] Electrowetting forces are optically mediated, and in this art, optoelectro This approach is publicly known as "fetting," and below, variations of this approach will be referred to by the corresponding abbreviation OEWOD. Examples of the format include US20030224528, US20150298125, US201 60158748, US20160160259, and Applied Physic These are disclosed in Letters 93 221110 (2008). In particular, these The first of the three patent applications is a microphone defined by the first and second walls. It includes a fluid cavity, the first wall is a composite design, and the substrate, photoconductive layer and insulating (dielectric) layer. Various microfluidic devices composed of layers are disclosed. In this one-sided embodiment, photoconductive Between the layer and the insulating layer, there are electrically insulated layers that are bonded to the photoactive layer, and their function is that of an insulating layer. An array of conductive cells that generates corresponding electrical wetting electrode positions. These are arranged. At these positions, the surface tension characteristics of the droplets are electrowet as described above. It can be changed depending on the field. Then the conductive cell collides with the photoconductive layer. It may be temporarily turned on by light. The usefulness of this approach lies in the arrangement of the electrodes. Although still somewhat limited, switching becomes much easier and faster. It has the advantage of allowing the droplet to move at a certain speed, and the actual droplet diameter. There are limitations regarding the extent to which the path can be altered.

[0004] Two-sided embodiments of this latter approach are presented by Pei at the University of California at Berkeley paper UCB / EECS-2015-119 This is disclosed in [publication name]. For example, a light on electrically biased amorphous silicon. An optoelectronic pattern is used to apply an optoelectronic pattern across the surface of Teflon® AF deposited on a dielectric layer. Using electrowetting, relatively large droplets with a particle size range of 100-500 μm are produced. The cell that enables the operation is described. However, in the example device, the dielectric The layer is thin (100 nm), and the photoactive layer is placed only on the bearing wall.

[0005] The entirety of our published application WO2018 / is incorporated herein by reference. In 234445, we provide optoelectrowetting to power the engine. This describes a device for manipulating microdroplets using optically mediated In electrowetting (OEWOD) devices, microdroplets contain walls. Thus, a microfluidic space is defined, for example, a pair of parallel spaces with a microfluidic space in between. It moves through the plate. At least one of the containment walls has a semiconductor layer embedded inside. The following "virtual" electrowet is generated by selectively irradiating a region. This includes what is called the ignition electrode position. It involves selectively illuminating a layer with light from a separate light source. This allows for the temporary generation of virtual paths for virtual electrowetting electrode locations. This allows for the movement of tiny droplets along the same path.

[0006] The entirety of our corresponding published patent WO2018 is incorporated herein by reference. / 234448 describes the use of this device as the operating part of a nucleic acid sequencer. It is being done. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] For the rapid screening and manipulation of biological samples containing cells the inventors have developed a device for applying a microdroplet method similar to that underlying the previously described sequencer of the inventors to apply.

Means for Solving the Problems

[0008] Therefore, according to the present invention, there is provided a device for manipulating and / or determining one or more characteristics for analyzing cells contained within a biological sample, the device comprising: a sorting component configured to separate cell-containing microdroplets from empty ones into a population of cell-containing first microdroplets; a microdroplet manipulation component configured to manipulate the first microdroplets using real or virtual electro-wetting electrodes, the microdroplet manipulation component comprising: a first zone configured to arrange the first microdroplets in an array for optical inspection and optionally introduce a reporter system into each first microdroplet by microdroplet merging; a second zone located within or adjacent to the first zone and configured to detect merged microdroplets within one or more detection windows; and optionally, a third zone capable of subdividing the microdroplets and isolating them for later recovery from the device; and an optical detection system configured to detect an optical signal from the merged microdroplets through one or more detection windows, the optical detection system being such that for the merged microdroplets, the signal results from an interaction between the reporter system and the cell or its expression product. a microdroplet manipulation component: a first zone configured to arrange the first microdroplets in an array for optical inspection and optionally introduce a reporter system into each first microdroplet by microdroplet merging; a second zone located within or adjacent to the first zone and configured to detect merged microdroplets within one or more detection windows; and a third zone located within or adjacent to the first zone and configured to detect merged microdroplets within one or more detection windows; and optionally, a third zone capable of subdividing the microdroplets and isolating them for later recovery from the device; and an optical detection system configured to detect an optical signal from the merged microdroplets through one or more detection windows, the optical detection system being such that for the merged microdroplets, the signal results from an interaction between the reporter system and the cell or its expression product. a third zone; and an optical detection system configured to detect an optical signal from the merged microdroplets through one or more detection windows, the optical detection system being such that for the merged microdroplets, the signal results from an interaction between the reporter system and the cell or its expression product. a third zone; and an optical detection system configured to detect an optical signal from the merged microdroplets through one or more detection windows, the optical detection system being such that for the merged microdroplets, the signal results from an interaction between the reporter system and the cell or its expression product.

[0009] According to another aspect of the invention, the device according to the present invention is used to extract fine particles from a biological sample. A method for manipulating and / or determining one or more characteristics of a cell type, comprising the following steps: The law is provided: By creating aqueous first microdroplets in an immiscible carrier fluid from biological samples, To create, and to create, which is believed to contain at least some cells of a specific cell type. thing; The first microdroplet is guided along the path using a real or virtual electrowetting electrode. This involves moving to at least one microdroplet inspection position; and The content of each microdroplet is analyzed using an optical detection system, and the amount of cells contained in each microdroplet is determined to be one-third of the total cells. The above characteristics are determined, and one or more of the above characteristics are: cell morphology, cell movement, or at least one of the cell membrane integrity characteristics, which can be determined by analysis. and.

[0010] According to another aspect of the present invention, the device according to the present invention is used in a biological sample A method for manipulating and / or determining one or more characteristics of a cell type, comprising the following steps: A method is provided: By creating aqueous first microdroplets in an immiscible carrier fluid from biological samples, There are, and it is believed that at least some of them contain cells of the cell type in question, to create and; The first microdroplet is directed along the path using a virtual electrowetting electrode at least Move to a single microdroplet merging position; Using a virtual electrowetting electrode, along the path to the microdroplet fusion site A second aqueous microdroplet containing a reporter system characteristic of the cell type whose properties are being investigated is moved. To cause; Combining the first and second microdroplets at the merging position to produce a merged microdroplet; Beauty The content of each merged microdroplet is analyzed using an optical detection system, and the relationship between the cell and the reporter system is analyzed. To detect optical signals characteristic of the interaction between them.

[0011] This method may initially include one or more initial steps of sorting, culturing, and droplet preparation. These initial steps may include one or more of the following: (a) From a collection of microdroplets including both cell-containing and empty microdroplets, cell-containing Separating the first microdroplet; (b) under conditions that induce cell proliferation and division before or after the initial step (a) is performed. Culturing a collection of microdroplets; (c) Applying electrowetting force to microdroplets from biological samples The process of generating a collection of tiny droplets by cleavage.

[0012] In some embodiments of the initial step (c), the microdroplets are immiscible carriers containing oil. The fluid contains growth medium components that have been cleaved, thereby continuing in the initial step (b) Prepare an emulsion that can be cultured. Alternatively, microdroplets can be mixed with air or another gas mixture. For example, they are cleaved into a mixture of carbon dioxide and nitrogen, and then cultured separately. In some embodiments, carrier fluids containing gases harmful to cell culture are periodically purged. This could be advantageous.

[0013] In one embodiment of the initial step (b), the culture of a group of microdroplets is performed in an emulsion. and the flow of non-miscible carrier fluids such as hydrocarbons or fluorinated oils, especially fluorinated oils. This is carried out in the presence of surfactants and Other additives may be optionally included. The oil also sustains cell proliferation during the culture stage. It contains low levels of nutrients and gases necessary for maintenance, and the application of this embodiment is, The nutrient content of microdroplets is replenished periodically or continuously by interfacial diffusion. In other words, the replenishment of nutrients and gases is achieved by the merging of secondary aqueous microdroplets containing these components. This can be achieved directly, for example, when the carrier fluid is air or an inert gas. be. In another embodiment, to provide a hypoxic environment for the cells, a specific dissolved gas is purged from the oil. do.

[0014] In one embodiment of the initial step (b), we use microdroplets to maintain cell proliferation. It was found that it is necessary to maintain optimal levels of specific atmospheric gases within the system. For example, this could cause harmful changes in the pH of the microdroplet medium. In one embodiment, in the initial step (b), the flow of the immiscible carrier fluid is one or more saturates It contains nitrogen, oxygen, or especially carbon dioxide at a sum level.

[0015] In another embodiment of the initial step (b), the content of the microdroplets is such that the microdroplets are held By applying electrowetting force at a specific location, stirring (stirred) is achieved. It is agitated. Appropriately, this is the type described below, for example. Optically mediated electrowetting force delivered by the OEWOD structure This is achieved by using [this method]. The inventors believe this approach also has broader utility. Therefore, in another generally applicable second aspect of the present invention, the content of the microdroplets is stirred. A method is provided for stirring, or if that is not possible, the method includes the following steps: Positioning microdroplets at the virtual electrowetting electrode location; and Applying an electromagnetic radiation source to the location and thereby creating a corresponding virtual electrowetting The electrodes are activated, and the electromagnetic radiation source moves around that location, causing the corresponding movement of the microdroplets. The corresponding stirring or agitation of its contents To generate associated electrowetting forces, characterized by causing them.

[0016] In some embodiments, the biological sample contains one or more male and / or female gametes. This method may include, as part of an in vitro fertilization workflow, male and / or This may further include manipulation and examination of female gametes.

[0017] For example, this instrument can be used to analyze male gamete cells such as human or animal sperm cells. It is possible to perform inspection, selection, and assay steps. In one example procedure, sperm cells The cytoplasmic sample is prepared from diluted semen and encapsulated in droplets. The droplets are then chipped. Load onto the surface and then examine using a bright-field microscope. Next, remove the droplets that do not contain gametes. Discard and retain any containing spermatozoa for examination. Once the gamete sample is ready, Once a sample is selected for analysis, a video of the gametes is taken along with still images. Optical detection system The pattern recognition algorithm applied to the output is based on kinetic, morphological, and nuclear morphological characteristics. This allows for the characterization of the gametes. The results of this characterization can be mapped to specific droplets. This droplet can then be removed for further processing. This includes on-chip assay steps such as the addition of a reporter reagent.

[0018] In another example, by encapsulating female gametes such as human or animal eggs, It is possible to fertilize the egg. Similar to male gametes, female gametes are encapsulated in droplets. It can be loaded onto a chip. Once placed on the device, the cells can be mo Fractures in the phology can be examined and assayed using a reporter reagent. After testing or assay, female gamete cells are subjected to any processing steps (e.g., via droplet movement). The embryonic epithelial cells are subjected to mechanical shearing by the application of the mechanical shearing or by the addition of further reagents. It can be used for removal.

[0019] In another example, male and female gametes were loaded onto a single microfluidic device. This process merges droplets containing two gametes together and binds them together. This is possible. For example, multiple male gamete droplets may merge with a single egg; between gametes Conventional interactions lead to fertilization and the generation of blastocyst-on-chip. In another example, a single selection Combined male gametes and a single selected and processed female gamete on-chip Combine and make them interact.

[0020] Another application involves recovering hermaphroditic gametes from a microfluidic chip and administering ICSI or IV. Combine them using conventional handling techniques known in this field, such as F.

[0021] In some embodiments, by the methods detailed above, or known in the art Blastocysts that can be formed by conventional means are encapsulated in droplets and cultured on-chip. It is also possible. On-chip culture uses the imaging and detection systems described below to determine the formation of cells. Allows for examination of blastocysts in between. Using the droplet merging procedure, the blastocyst environment is buffered with salt. By adding extra materials such as nutrients, proteins, and extracellular matrix materials It can be controlled. During blastocyst formation, a sample of cells is removed from the blastocyst for further analysis. To retrieve them, techniques such as laser microscopy are often used. Desirable. In some embodiments, the blastocyst is transported to a droplet manipulation zone. This manipulation zone The line is the pillar, then the physical limit between the electrowetting plate, or PCT / Electrowetting plate as described in EP2019 / 062791 This can include physical features on the microfluidic chip, such as wedge-shaped changes in the gaps between them. The disclosure is incorporated herein by reference. Once the blastocysts are loaded into the operating zone, Once fixed, it is effectively secured. Then, laser microscopy can proceed. The process of removing a portion of the blastocyst is well documented in the literature. When a portion of the droplet is removed, the droplet division procedure described herein is performed on the blastodisc. It can be used to separate sample portions from cells. Distribution of material between two droplets after division. Repeated division and recombination operations, as well as machine-visual inspection, reveal the blastocyst and sample parts. It is possible to verify that the parts have been correctly separated. After separation, the blastocyst sample portion Further analysis is performed through genetic testing, including polymerase chain reaction or DNA sequencing. It can be recovered for analysis.

[0022] Appropriately, the electromagnetic radiation source used in this method is the second electromagnetic radiation described below. This includes a rapidly flashing rotating light source that depicts a circular path within or around the location. In another embodiment, the movement of the light source may include a path of one or more lateral movements. In one manifestation, the position is defined by an area with a diameter of at least 0.5 microns, and the motion Its motion is circular, radial, or a mixture of both. Its motion is due to the interaction of the contents of the microdroplets. The radial direction is used to produce the corresponding centrifugal mixture.

[0023] The reporter system that can be introduced into the first microdroplet in step (4) is, in principle, a biological It is characteristic for assaying the presence of a given cell type or cellular behavior in a scientific sample. Or it could be any system that can be used. Such reporter systems include, for example, against enzymes, proteins, or antibodies expressed by cells of the desired cell type Examples include selective reporter genes, cell surface biomarkers, or reporter molecules. The reporter system of the relevant class contains the relevant material expressed by the cell being searched. It may be a second reporter cell that responds to the presence of the cell. Many such assays are known, And suitable candidates for use are often obvious to those skilled in the art. Furthermore, multiple Different reporter systems are introduced into the first microdroplet by the merging of one or more second microdroplet types. By doing so, this method allows for a range of different settings related to diverse and different characteristics and behaviors. It is understood that this can be multiplexed to perform parallel and simultaneous searches on the type. It can be done. [Brief explanation of the drawing]

[0024] [Figure 1] Refer to Figure 1 for an example of a device and its associated workflow. [Figure 2] Figure 2 shows a cross-sectional view of an exemplary device, an oEWOD structure having a diameter of 120 μm (e.g., 80-120 μm) in an unconfined state, suitable for rapid handling of aqueous microdroplets 2 emulsified in a fluorocarbon oil having a viscosity of 5 centistokes or less at 25°C. [Modes for carrying out the invention]

[0025] The method of the present invention, its various steps, and the first substep are described below. This can be conveniently carried out using the analytical device. Applicable optical detection systems Examples are described below. In some embodiments, this device includes: A saw for separating cell-containing microdroplets into empty ones and then into a cluster of cell-containing first microdroplets. Ting component; The first microdroplet is then manipulated using a real or virtual electrowetting electrode. A microdroplet manipulation component for which the microdroplet manipulation component includes the following: nt: The method includes means for introducing a reporter system into each first microdroplet by microdroplet merging, Zone 1; The merged microdroplets are then detected within one or more detection windows in the first zone or Zone 2, located adjacent to and The aforementioned reporter system, bright-field microscope, dark-field microscope, chemiluminescence detection means, and Förster Phase with cells or their expression products selected by resonance energy transfer detection means or fluorescence detection means An optical detection system that detects optical signals from merged microdroplets caused by interaction.

[0026] The sorting component used in such embodiments includes one or more cells. A larger collection of microdroplets (hereinafter referred to as "filled microdroplets"), some of which are empty. This is a means of separating from the group. Depending on the type of sorting component selected. Therefore, depending on whether the microdroplets are filled or empty, there are two different types of microdroplets. It is directed towards or downward to one of the fluid pathways or receiving locations. In the embodiment, access to one or the other of these is a fraction of each microdroplet in the analysis window. Controlled by the partition or operation of an electromechanical gate that acts in response to the analysis. Another implementation Morphologically, sorting involves optically mediated electrowetting of the responding sample within the analysis window. Applying a suction force to a flow of microdroplets draws selected droplets into a holding area or array. This is achieved by allowing the droplets to be drawn in. The rejected droplets then remain in the airflow. Then, it can be discarded. In another embodiment, the sorting determination is an optical phenomenon. For example, based on bright-field microscopy, or on cell-related optical properties, such as fluorescent tags. or by detecting the presence of a marker. In another embodiment, sorting is temporary An electric field is applied to the analysis window, and each minute droplet is subjected to one of two different paths on either side of the partition. This can be achieved by a dielectrophoretic method that sequentially deflects in one direction. The sorting component consists of a first microfluidic channel that terminates within the analysis chamber, and its small At least one of the two is connected to the downstream analysis chamber that carries away the first microdroplet. Two microfluidic channels, a light source for illuminating the analysis chamber, and each illuminated microfluidic channel within the analysis chamber. A bright-field microscope or fluorescence detector for acquiring data from a droplet, and two second channels One of them is an OEWOD structure capable of acting to guide a microdroplet, and a micro The results depend on the identification algorithm applied to the data received from the mirror or fluorescence analyzer. It includes a microprocessor adapted to respond to and operate the structure.

[0027] In one embodiment, the device is an integrated component of the device itself It is either a sorting component, or preferably a sorting component, before or after the sorting component. Further including culture components that are placed separately after the culture component. Here, Microdroplets are retained, but any cells contained within them are stimulated to divide and proliferate. They are cultured in such a way. Preferably, the culture components are microdroplets under optimal culture conditions. In terms of type, a container that can be kept at temperatures above 25°C (for example, 25-40°C) for 1 hour to 1 week. and an inlet for introducing microdroplets into it. In one embodiment, the culture component The system includes a thermostat-controlled heater and, optionally, a device charge / discharge cycle. The device further includes a timer that controls the cycle. In one embodiment, the device contains non-mixed The device contains an emulsion of microdroplets in a harmonious carrier fluid, and the carrier fluid changes over time. It further includes entrances and exits that can pass through it, which allow it to be replaced by a different system. In one embodiment, the immiscible carrier fluid is HFE7500, HFE7700, or FC These are fluorocarbon oils such as -40. Such oils maintain the stability of microdroplets. Furthermore, in order to maintain low levels of nutrients and gases necessary for sustaining growth, the interface activity It further contains appropriate properties and other additives.

[0028] In one particularly useful embodiment, where the weight ratio of aqueous microdroplets to oil is low, the microdroplets over time Both tend to contract, and this phenomenon can lead to a loss of reactivity within the microdroplet. One way to counteract the effect is to use hydrated oil. Generally, the above oil Since the oil does not have a high capacity to dissolve water, hydration occurs in the oil phase with water or an aqueous buffer. This is adequately achieved by generating liquid micelles or secondary microdroplets. This buffer is The microdroplets may have the same or different composition as the microdroplets themselves. Several implementations In this state, these micelles or secondary microdroplets contain up to five times the salt content of the microdroplets themselves. They may be made to contain glycerin, and may optionally contain glycerin. Typically, these micelles and two The next microdroplet is an order of magnitude smaller.

[0029] In one embodiment, the container uses optically mediated electrowetting forces It has multiple positions where minute droplets can be placed and stirred to agitate their content. It further includes a surface.

[0030] The device is further integrated with the device itself, or with aliquots of biological samples. The method includes means for creating an emulsion of microdroplets in an immiscible carrier fluid. Includes either a sample preparation component or one that is positioned separately upstream of the nutrient component. nothing.

[0031] This sample preparation component cleaves microdroplets from biological samples into a carrier fluid. The cutting means includes a cutting means for which an electro-moistened stretching force is applied to a biological sample. It includes at least one position. In one embodiment, the cutting means includes: A first electrowetting site adapted to receive biological samples; The positions of the first and second electrowetting electrodes are directional electrowetting. To define a pathway through which microdroplets cleaved from the sample can be transported using force. At least one second electrowetting position is positioned; It is positioned at the first electrowetting location, and the electrodes and associated AC electricity AC consists of either a circuit or a semiconductor zone activated by electromagnetic light collisions. Drive circuit and It is positioned at the first electrowetting position and electrostatically coats the surface of the biological sample. A DC charging circuit adapted for electrical charging.

[0032] In one embodiment, the cutting means is a drive circuit and a charging circuit which are appropriately AC and DC circuits. The system further includes a control circuit for switching between the two. In another embodiment, the cutting means is This further includes an analyzer for analyzing the content of each microdroplet generated from a physical sample. In this respect, biological samples are those derived from blood, plasma, sputum, urine, or tissue biopsy. It may be the formation of any aqueous substance such as a certain quality. Further information on suitable cutting methods is available at This can be found in our concurrently pending application EP18201162.7, which is of interest to the reader. The microdroplet cutting method related to the cutting means forms the criterion for performing the first step (c) described above. It will be easy to understand that this is possible.

[0033] The first microdroplets generated by the sorting component are used for subsequent manipulation. Looking at the microdroplet manipulation components used, this appropriately corresponds to the first zone, The second zone and the first microdroplet are subjected to aerodynamic and / or electrowetting forces. And connected to one or more microfluidic paths driven along them, A microscopic detection system comprising an optical detection system consisting of an actual or virtual electrowetting electrode. This is a small fluid chip. Preferably, the electrowetting electrode is virtual, and one or more It is established in the position within the OEWOD structure above. Generally, this manipulates the microdroplets in this method. This is a method for creating these OEWOD structures, and in one embodiment, these OEWOD structures include the following: The first composite wall includes the following: First board A first transparent conductive layer on a substrate, the first transparent conductive layer having a thickness in the range of 70 to 250 nm. ; The photoactive layer was activated by electromagnetic radiation with wavelengths of 400-850 nm on the conductive layer. The photoactive layer has a thickness in the range of 300 to 1500 nm. and A first dielectric layer on a photoactive layer, the first dielectric layer having a thickness in the range of 30 to 160 nm. ; The second composite wall includes the following: Second substrate; A second conductive layer on the substrate, having a thickness in the range of 70 to 250 nm, body layer and Optionally, a second dielectric layer on a second conductive layer having a thickness in the range of 30 to 160 nm The second dielectric layer The exposed surfaces of the first and second dielectric layers are spaced 20 to 180 μm apart. a second composite wall that defines a microfluidic space adapted to contain minute droplets; A voltage is provided between the ends of the first and second composite walls that connect the first and second conductive layers. , A / C source; To induce the corresponding virtual electrowetting position on the surface of the first dielectric layer Higher energy than the band gap of the photoactive layer, adapted to collide with the photoactive layer. - at least one electromagnetic radiation source having; and The arrangement of virtual electrowetting positions is changed, thereby moving the microdroplets. To generate at least one electrowetting path that can be opened, light A means for manipulating the collision points of electromagnetic radiation on the active layer.

[0034] In one embodiment, the first and second walls of these structures have a microfluidic space in between. It is transparent. Alternatively, the first substrate and the first conductor layer are electromagnetic radiation sources (e.g., multiple lasers). - Allows light from a beam, lamp, or LED to collide with the photoactive layer. It is transparent. Alternatively, the second substrate, the second conductor layer, and the second dielectric layer can be obtained with the same objective lens. It is transparent so that it can be seen. In another embodiment, all of these layers are transparent.

[0035] Appropriately, the first and second substrates should be made of mechanically strong material, such as glass, metal, or enamel. Made from engineering plastic. In one embodiment, the substrate has a certain degree of flexibility. It can have. In yet another embodiment, the first and second substrates are 100-1 It has a thickness in the range of 000 μm. In some embodiments, the first substrate is silicon, It is composed of fused silica and one of glass. In some embodiments, the second The base material is composed of quartz glass and one of the following types of glass.

[0036] The first and second conductive layers are located on one surface of the first and second substrates, and typically It has a thickness in the range of 70 to 250 nm, preferably 70 to 150 nm. One embodiment So, at least one of these layers is a permeable material such as indium tin oxide (ITO). From bright conductive materials, very thin films of conductive metals such as silver, or conductive polymers such as PEDOT These layers are fabricated. These layers are formed as a series of discrete structures such as continuous sheets or wires. Alternatively, the conductive layer may be a conductive material in which electromagnetic radiation is directed between the mesh gaps. A mesh made of material is also acceptable.

[0037] The photoactive layer generates localized charge regions in response to stimulation from the second electromagnetic radiation source. It is appropriately constructed from available semiconductor materials. For example, thickness in the range of 300 to 1500 nm. Examples include a hydrogenated amorphous silicon layer having properties. In one embodiment, the photoactive layer is It is activated by the use of visual light.

[0038] The photoactive layer in the case of the first wall and the optional conductive layer in the case of the second wall are typically 30~ It is coated with a dielectric layer with a thickness in the range of 160 nm. The dielectric properties of this layer are: Preferably, it contains a high dielectric strength of 10^7 V / m and a dielectric constant >3. Preferably, It is as thin as possible, in line with avoiding dielectric breakdown. In one embodiment, the dielectric layer This is selected from alumina, silica, hafnia, or a thin non-conductive polymer film. .

[0039] In another embodiment of these structures, at least the first dielectric layer is preferably both Desired microdroplets / carrier fluid / surface at various virtual electrowetting electrode positions It helps to establish a contact angle, and furthermore, the microdroplet content adheres to the surface, microliquid To prevent the droplets from decreasing as they move through the tip, a fouling-resistant coating is applied. If the second wall does not contain the second dielectric layer, the second antifouling layer is the second conductor. It may be applied directly to the layer. For optimal performance, the anti-fouling layer should be air-liquid-surface at 25°C. When measured as a three-point interface, the microdroplet / carrier fluid / surface contact angle is 50-170°. It should help to establish. In one embodiment, these layers have a thickness of less than 10 nm. They have, and are typically monolayers. In another embodiment, these layers have hydrophilic groups, for example acrylate esters substituted with alkoxysilyl, for example, methyl methacrylate or It consists of polymers of its derivatives. One or both of the stain-resistant layers ensure optimal performance. Therefore, it is hydrophobic. In some embodiments, in order to provide chemically compatible crosslinking, 20 An interstitial layer of silica with a thickness of less than nm is interposed between the antifouling coating and the dielectric layer. It is possible.

[0040] The first and second dielectric layers, and therefore the first and second walls, have a width of at least 10 μm. The microfluidic space is a micro-air, preferably in the range of 20 to 180 μm, and contains minute droplets. The space between them is defined. Preferably, the microdroplets themselves are defined before they are included, and the width of the microdroplet space is defined. It has an intrinsic diameter that is more than 10%, and preferably more than 20%. By this means, Upon entering the tubing, the microdroplets are compressed, for example, through improved microdroplet merging capabilities. This improves electrowetting performance.

[0041] In one embodiment, the first and second dielectric layers are hydrophobic coatings such as fluorosilane. It is coated with a special coating.

[0042] In another embodiment, the microfluidic space maintains a predetermined distance between the first wall and the second wall. Includes one or more spacers for holding. Spacer options include optical patterns Beads or pillars, ridges generated from the intermediate resist layer produced by the finishing process. Examples include using a deposited material such as silicon oxide or silicon nitride. A ferrule may be made. Alternatively, a flexible plastic with or without adhesive coating may be used. A spacer layer can be formed using a film layer containing a stick film. Using various spacer shapes, with a narrow, tapered channel defined by the pillar line. A channel, or a partially enclosed channel, can be formed. Careful design is required. Therefore, these spacers can be used to help deform the microdroplets, and then, It is possible to perform effects on the division of small droplets and deformed microdroplets. These spacers are used to physically separate the zones of the chip and prevent cross-contamination between droplet clusters. This prevents staining and promotes the correct direction of droplet flow when the tip is loaded under hydraulic pressure. It is possible.

[0043] The first and second walls are biased using an A / C power source attached to the conductive layer. They provide a voltage potential difference between them, which is appropriately in the range of 10 to 50 volts.

[0044] These OEWOD structures typically have a wavelength of 400-850 nm, preferably 660 nm. A second electromagnetic field with wavelengths in the range and energies higher than the band gap of the photoactive layer It is used in conjunction with a radiation source. Preferably, the photoactive layer is used when the incident irradiation intensity is 0. 0.1~0.2 Wcm -2It is activated at the virtual electrowetting electrode position. In one embodiment, the electromagnetic radiation source is located on the photoactive layer, which is also pixelated, and corresponds to the photoexcitation region. The pixels are generated to produce the virtual electrowet. The tip electrode position is induced on the first dielectric layer.

[0045] When an electromagnetic radiation source is pixelated, it is illuminated by light from an LED or other lamp. Using reflective screens such as digital micromirror devices (DMDs), directly Alternatively, an electromagnetic radiation source is appropriately supplied. This creates a virtual electrical wetting electrode position. This allows highly complex patterns to be rapidly created on the first dielectric layer and then destroyed. This allows for the precise application of electrically controlled wetting forces to microdroplets, essentially creating any desired effect. This makes it possible to precisely control the aircraft along a virtual path. This is because the chip can control multiple paths. This is particularly advantageous when it is necessary to manipulate thousands of such tiny droplets simultaneously. The electrowetting path is a virtual electrowetting path on the first dielectric layer. It can be seen as being constructed from a continuum of electrode positions.

[0046] The collision point of the electromagnetic radiation source with the photoactive layer can be any convenient shape, including conventional circular or annular shapes. This can be done. In one embodiment, the form of these points corresponds to the form of pixelation. Therefore, it is determined, or alternatively, it is completely determined by the form of the microdroplet that enters the microfluidic space. Or partially corresponding. In one embodiment, the collision point, and therefore the electrical wetting electrode position, is crescent-shaped. It may be shaped and oriented in the intended direction of travel of the microdroplets. Preferably, it is electrically The wetting electrode position itself is smaller than the surface of the minute droplet adhering to the first wall, and the droplet and surface dielectric are This provides the maximum electric field strength gradient across the contact line formed between them.

[0047] In one embodiment of the OEWOD structure, the second wall is affected by the same or different electromagnetic radiation sources. The invention also includes a photoactive layer that enables the induction of virtual electro-wetting electrode positions on the dielectric layer 2. The addition of the second dielectric layer causes a transition of the wet edge of the microdroplets from the top to the bottom surface of the structure. This allows for the application of more electrowetting force to each microdroplet.

[0048] A first zone forming part of the device introduces a first microdroplet in one embodiment. An inlet for and an outlet attached to the second zone by an electrowetting path. It is a retaining reservoir equipped with a port. The first zone is a port for introducing a reporter system. The reporter system further includes, in one suitable embodiment, a first microdroplet A second aqueous microdroplet containing a reporter system designed to identify the properties of the cells it contains. This is a second entry point for introducing the first microdroplet. In one embodiment, the reservoir holds the first microdroplet. It can hold and further include an array of positions on which a second microdroplet is driven. This process causes some degree of merging of the first and second microdroplets. Then, The merged first / second microdroplets (hereinafter referred to as "merged microdroplets") are then detected by the reporter system. After interacting sufficiently with the cells, they then undergo a second process via electrowetting. It can be held in that position until it generates the optimal optical signal to be transported to the zone. In some cases, it is desirable to monitor the growth of the optical signal using time-resolved measurements. In the implementation configuration, a single zone encompassing the roles of both the first and second zones is, for example, above It is used by detecting the merging microdroplets at the merging location mentioned above.

[0049] The second zone preferably includes one or more detection windows through which an optical detection system is used. Then, the merged microdroplets can be analyzed. In one embodiment, the second zone is This is the transparent part of the pu. In another embodiment, the optical detection system is a reporter system and a cell or its emission Designed to detect optical signals from microdroplets arising from interactions with the actual product. Therefore, the optical detection system should be able to detect bright-field microscopes, dark-field microscopes, and chemiluminescence. Means for detecting Förster resonance energy transfer, or means for detecting fluorescence A means is selected for this purpose. In one embodiment, the detection system is selected for the combined microdroplets and / or It receives a signal from one of the detectors, for example, in the form of a visual display or count. This also includes a light source for illuminating a microprocessor that provides data to the user in a controlled manner. In one embodiment, the microprocessor controls the performance of the sorting component, the first zone The introduction rate of the first microdroplet into the nucleus, and the response to the signal detected by the optical detection system. To control one or more of the coalescence rates of the first and reporter system-containing microdroplets, Further adapted by a back loop.

[0050] A special advantage of instruments using the oEWOD structure for droplet manipulation is that it focuses on the sample. The instrument has a built-in optical addressing system. (Required for oEWOD control) Along with illumination, by multiplexing and demultiplexing the excitation light and emission light necessary for light detection, This allows many optical functions to be integrated into a single, simpler, and lower-cost assembly. It is possible. For example, long-path dichroic mirrors can be used to remove light from an assembly. By using multiplexing, it becomes possible to divert light from the operating column and to create a high-sensitivity detection camera. Another embodiment uses two dichroic mirrors. The first mirror is from the lamp To multiplex the fluorescence excitation light, a second mirror is used to multiplex the fluorescence emission. This requires more advanced lighting schemes such as time-resolved Förster resonance energy transfer. In this embodiment, in order to apply a time-dependent and spatially changing lighting pattern, It is preferable to employ the same structured lighting system that addresses the oEWOD operation pattern. Similar to using dichroic mirrors for these multiplexing operations, distributed mirrors Elements such as routers, dispersion lenses, or diffraction gratings can be used, depending on the application. Therefore, structured illumination systems implement temporal multiplexing, which is used to rapidly switch between excitation sources. It is preferable to do so.

[0051] In some embodiments, the device extracts microdroplets from the first and / or second zones. It further includes a third zone for collection. Here, the first microdroplet is, for example, more detailed It may be subdivided and isolated for subsequent recovery from the instrument, or for confirmatory analysis. In one embodiment, the third zone is connected to a temporary storage container, and is one of the zones from the second zone. It consists of multiple exit ports. Using the oEWOD transport mechanism, the first And by replenishing the second zone, a large number of microdroplets can be sequentially collected. This makes it possible to collect multiple droplets separately from a single port.

[0052] Similar to the optically mediated manipulation of fluids within an OEWOD structure, the device also allows for various By selectively applying hydraulic pressure to the inlet and outlet ports, the flow inside the device is controlled. This may include a network of pumps and valves for operating it. Preferably, this network The workpiece consists of a two-position valve connected to each outlet, and a set of pressure sources (Po) connected to the same valve. It includes a pump, a collection container, and a reservoir. By changing the configuration of each valve, Positive or negative pressure can be applied to the device via the reservoir and collection container. Therefore, material inflow into the device, material outflow from the device, or device This causes the influx of materials from within.

[0053] The devices and related methods of the present invention described above have many useful applications. Below, We will describe some application examples and related workflows.

[0054] One application example of the disclosed device and method is in the development of genetically modified cell lines. be.

[0055] In this application, target cells are optically mediated by the sample preparation components. Encapsulated in a first microdroplet via a cutting means based on lectrowetting The transfection reagent (e.g., modified lentivirus) is placed in a separate second microliquid. It is encapsulated in a drop.

[0056] Next, the first and second microdroplets in the merge operation described above are OEWOD deba They merge on the chair, forming merged microdroplets, and transfect target cells. Expose to the reagent. Cells in the merged microdroplets are divided between droplets. The cells are subjected to a cycle of blending and splitting operations, and the culture medium surrounding them is serially diluted. The culture medium is replaced with fresh medium, depleting substances are replenished, and any cellular excretion accumulated in the droplet is removed. Remove an object.

[0057] For example, microscopic examination during the assay can be used to track the position of each cell in a population of microdroplets. This allows for the identification of cells of common ancestry for sorting purposes. This ensures the monoclonal nature of the cultured cell population.

[0058] During the described process, freeze-thaw cycles, which are known to reduce cell viability, Because there are no harmful processes of distribution, manual manipulation, or repeated long-term passaging, cell retention is also This increases compared to conventional cell development methods. Similarly, the cells remain encapsulated in the droplet. This eliminates the possibility of losing clones to the surface of liquid processing equipment. Furthermore, non-viable cells Cells can be discovered early in the process and replaced by living cells instead.

[0059] Once it was determined that the cells in the fused microdroplets had been cultured for a sufficient length of time, Then, a third reagent, including the reporter assay, is introduced into the OEWOD device, targeting cells. It is combined with a microdroplet containing [the substance]. The results of the reporter assay are, for example, the detected fluorescence, [the substance]. It can be measured according to luminescence, or Förster resonance energy transfer.

[0060] Based on the results of the reporter assay, one or more primary subsets of cells may be discarded. and it may be possible to further grow one or more second subsets of cells. The sample is cultured Cells were recovered from a subset of acquired cells and removed from the chip for further off-chip analysis. The cultured subsets are distributed into a well plate, for example, a standard 1536-well plate. The remaining progenitor cells are retained for further culture on the chip.

[0061] Next, the recovered samples undergo one or more off-chip analyses (e.g., DNA sequencing, RN). (For use in sequencing, PCR analysis, gene profiling, and microarray measurement) Based on the results of the off-chip analysis, a further subset of on-chip cells is selected. They can then be collected and further cultured.

[0062] Another application of the present invention is screening cells for immune function.

[0063] After immunization with antigens such as toxins or biomarkers characteristic of the disease, B cells, T cells... Alternatively, samples of natural immune cells such as dendritic cells are taken from organisms such as mice, humans, or primates. They can be collected from the surrounding tissues, lymph, and blood cells of the host organism. To separate the cells and other components, they are processed and purified. This involves incision, centrifugation, This can be achieved by a mixture of immunoprecipitation, filtration, and dialysis methods.

[0064] The purified immune cells are encapsulated in microdroplets and loaded onto the OEWOD device. Next, an immunoassay reagent, a FRET reporter, or a reporter cell line is used. Which reagent is introduced into the microdroplets containing the target cells in the first assay? As described above, this is performed by generating a second microdroplet of the reagent and carrying out the merging operation. It is possible.

[0065] The results of the first assay are examined, for example, by optical detection or microscopy, and antibodies Proteins like these are excreted by target cells in response to immunoassay reagents. To determine whether to proceed, based on the results of the first assay, the microdroplets containing target cells are subjected to a assay. One subset can be discarded from the device, and another subset can be retained for further testing. It will be done.

[0066] Next, a second reagent, such as an off-target reporter, is used in the second on-chip assay. In this case, the remaining cell-containing microdroplets may be introduced in the same manner as the others. The result of the second assay The results were measured using optical techniques such as fluorescence spectroscopy, and the second round of microdroplet assays were performed. This guides the selection and disposal of the sample. The above process uses a series of different reporter assays. Query / screen target cells and identify on-target, off-target, and irrelevant targets. This can be repeated to measure the target cell's response.

[0067] As used herein, the term "on target" refers to the end tissue or antigen. The target cells are producing a response antibody. For example, the on-target could be cancerous tissue. Where used herein, the term “off-target” means that an undesirable effect is observed. This refers to tissue that is close to or expected to be cancerous. For example, off-target refers to tissue that is close to or associated with cancerous tissue. It may be healthy tissue. Where used herein, the term “irrelevant target” It is not expected to have biological interactions with antibodies, but for example, a large amount at a useful end Substances that may negatively affect the accuracy of assay results through antibody binding or confounding measurements. It refers to.

[0068] Based on the results of the screening assay, select the final subset of cells. Next Then, the remaining cell-containing microdroplets, including the final subset, are lysed with the selected lysis reagent and cD By introducing it into an NA synthesis reagent, a library of genes currently expressed in target cells is obtained. Formation. The target cells are harvested outside the chip and observed in a phenotypic assay on the chip. It will be subjected to a gene assay to identify the coding DNA involved in movement.

[0069] Another application of the present invention is the functional properties of drugs, including immunomodulatory agents and drugs for tumor suppression. And screening for effectiveness.

[0070] In this application, a panel of drug-targeted cells is encapsulated in a first microdroplet. It is then loaded onto the OEWOD device. It includes a panel of drug compounds for testing. A second set of microdroplets is also loaded.

[0071] The dose measurement panel consists of a panel of microdroplets containing a range of dilutions of each drug compound. To produce it, it is formed by merging and splitting operations performed on a second microdroplet. The drug compound is encapsulated within the vesicle, even if it is in the formation of microbeads. It may also be expressed by producing cells encapsulated within a droplet.

[0072] The drug dosimetry panel is introduced into target cells by a compounding procedure; drug dosimetry Comprehensive pairwise combinations between the first cell containing the panel microdroplets and the microdroplets. Seth ensures that the entire panel is repeatedly exposed to all cell types. T cells (e.g., T killer cells or macrophages) also play a role in regulating immune responses. The effects of the drug will be tested, and detailed cross-sectional comparisons of immune responses in the presence of different tissues will be performed. Therefore, it can be introduced along with a drug panel and target cells.

[0073] The response of target cells to a drug panel can be assessed, for example, by microscopy, fluorescent reporter staining, or It can be monitored by a reporter assay. Using the results of the screening process This provides information on the efficacy of the test drug under various cell and effector cell conditions. It is possible.

[0074] Another application of the present invention is to induce differentiation of target stem cells.

[0075] In this application, target stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells) Or hematopoietic stem cells are encapsulated in a first microdroplet, and then in the OEWOD device. It will be loaded on top.

[0076] For example, growth factors, environmental stimulants, cell-to-cell signaling compounds, and moles A panel of control reagent compounds, such as fogen, is encapsulated in a second microdroplet, and also, It is loaded onto the OEWOD device.

[0077] A subset of the first microdroplets exposed the stem cells contained in the first microdroplets to the reagent. Therefore, to promote the differentiation of stem cells along the target pathway, a second micro-container containing a control reagent is used. It merges with the droplet.

[0078] The stem cell differentiation process can be analyzed, for example, through microscopic imaging, detection of phenotypic reporter compounds, and This is monitored by performing a Porter assay. Differentiated cells in merged microdroplets The cells are recovered from the OEWOD device via a dispensing process for further culture or processing. It is possible.

[0079] Another application of the present invention is in the controlled formation of organoid structures. .

[0080] In this application, organoid progenitor cells (e.g., tumor cells or stem cells) are the first It is encapsulated in a microdroplet and then loaded onto an OEWOD device. For example, Regulatory reagents such as growth factors, environmental stimulants, intercellular signaling compounds, and morphogens. The panel is encapsulated in a second microdroplet and loaded onto the OEWOD device. It will be done.

[0081] A subset of the organoid progenitor cell population contained in the first microdroplet is an organoid. To promote the formation of tissue structures, the tissues are exposed to a control reagent through a mixing procedure. The organoids formed in this way are stored in a dedicated area on the OEWOD device. Nutrients and any other necessary growth media can be supplied via a droplet merging operation. The organoids stored on the chip in this format are described in relation to the exemplary application described above. Thus, it can be used in drug screening assays.

[0082] Another application of the present invention is in CRISPR-Cas9 gene modification screening. That is the case.

[0083] In this application, target cells are encapsulated in a first microdroplet, and then OEWO A second set of microdroplets containing a panel of gRNA pairs is loaded onto the D device. The gRNA pair panels are then loaded onto the device. The gRNA is spotted onto the target region on the device surface, and then microdroplets are placed on top of them. This may include a preparatory step of rehydrating the area containing the panel by passing it through.

[0084] This is because gRNA binds to microbeads, and these beads are spotted in the fluid. This may involve the formation of a bead prep process that is freeze-dried on the surface.

[0085] The gRNA is introduced into target cells via a merge operation, which then leads to the target cells Programmable restriction enzymes (e.g., Cas9) and gene modification in target cells The gRNA is incorporated along with the reagents necessary to induce the mutation.

[0086] Cells in merged microdroplets are divided between droplets, and the medium surrounding the cells It is subjected to a cycle of merging and splitting operations, which are exchanged through continuous dilution.

[0087] By tracking the position of each cell within a collection of microdroplets, cells of common ancestor origin can be sorted. This makes it possible to identify cells for research purposes, and increases the monoclonality of cultured cell populations. The number of people who own it will also increase.

[0088] It was determined that the cells in the fused microdroplets were cultured for a sufficient period of time. Then, a third reagent, including a reporter assay, is introduced into the OEWOD device, and the target cells are activated. It is combined with the contained microdroplets. The results of the reporter assay are, for example, the detected fluorescence, chemistry It can be measured according to luminescence or Förster resonance energy transfer.

[0089] Based on the results of the reporter assay, one or more primary subsets of cells may be discarded. and it may be possible to further grow one or more second subsets of cells. The sample is cultured Cells were recovered from a subset of acquired cells and removed from the chip for further off-chip analysis. The cultured subsets are distributed into a well plate, for example, a standard 1536-well plate. The remaining progenitor cells are retained for further culture on the chip.

[0090] Next, the recovered samples undergo one or more off-chip analyses (e.g., DNA sequencing, RN). (For use in sequencing, PCR analysis, gene profiling, and microarray measurement) Based on the results of the off-chip analysis, a further subset of on-chip cells is selected. They can then be collected and further cultured.

[0091] Refer to Figure 1 for an example of a device and its associated workflow.

[0092] Fluid inlet 1 is an emulsion of a mixture of empty cell-containing first microdroplets in fluorocarbon oil. Add 2. Next, these first microdroplets are formed by an OEWOD structure (not shown). They are then moved to sorting zone 3, where they are sorted by optical means or other sorting methods as described above. The sorting mechanism sorts them into empty ones 4 and those containing cells 5. Then the cells Each of the contained microdroplets 5 is also moved to the confluence zone 8 by the OEWOD structure, there They are maintained for a specified period under conditions that promote cell proliferation and division within each of them. At the end of this period, the second inlet 6 is a fluorescent reporter selective for the target cell type 7. A second microdroplet containing the system is added, and then these are combined in confluence zone 8 with the first microdroplet containing cells. It merges with droplet 5 to form a merged microdroplet 9. Then, the merged microdroplet 9 becomes O The EWOD structure transfers the signal to the optical window 10, where the fluorescence signal characteristic of the reporter system is transferred. An optical detection device 11 consisting of an LED light source, a photodetector, and a microprocessor is used for detection. The optical detector 11 is partially combined with the optical operation projector 12.

[0093] Figure 2 shows a fluorocarbon oil with a viscosity of less than 5 centistokes at 25°C, mixed with milk. Suitable for rapid handling of conditioned aqueous microdroplets 2, in an unconfined state, 120 μm (e.g., A cross-sectional view of an exemplary device is shown, illustrating an oEWOD structure with a diameter of 80-120 μm. This is a transparent layer of conductive indium tin oxide (ITO) 15 with a thickness of 130 nm. Upper and lower glass plates (13 and 14) coated to a thickness of 500 μm each ) includes. Each of the 15 is connected to the A / C source 16, and the ITO layer on 14 is grounded. 14 is coated with a layer of amorphous silicon 17 with a thickness of 800 nm. 13 and Each of the 17s is coated with a 160 nm thick layer of high-purity alumina or hafnia 18. Next, trichloro(1H,1H,2H,2H-perfluorooctyl)silane 19 The layer is covered with an interstitial layer of silicon dioxide supporting it, resulting in a hydrophobic surface. 13 and 17 are designed so that the microdroplets undergo some degree of compression when introduced into the device. , spaced 80 μm apart using spacers. Reflective image illuminated by LED light source 20 The image on the rendering screen is generally placed below 14, and is 0.01 Wcm. -2 Visible light (wavelength 66 0 or 830 nm is emitted from each diode 21 and passes through 14 and 15. It can be made to collide with 17 by propagating in the direction of numerous upward arrows.

[0094] At various points of the collision, the photoexcitation region of charge 22 corresponds to the electrowetting At position 23, a modified liquid-solid contact angle is generated at 17, which is induced at 18. These improved properties are necessary for propelling the microdroplet 2 from one point 23 to another. It provides capillary force. 20 is brought to a specific point in time by a pre-programmed algorithm. It is controlled by a microprocessor 24 that determines which of the two 21s (I) is irradiated. .

Claims

1. To manipulate and / or determine one or more properties of cells contained within a biological sample A device that includes the following: The cell-containing microdroplets are configured to separate from empty droplets into a group of first cell-containing microdroplets. The sorting components that were created; We manipulate the first microdroplet using a real or virtual electrowetting electrode. A microdroplet manipulation component configured as follows, comprising: -Nent: For optical inspection, the first microdroplets are placed in an array, and optionally, each microdroplet is merged. A first zone configured to introduce a reporter system into the first microdroplet; Located within or adjacent to the first zone, and merged within one or more detection windows A second zone configured to detect microdroplets; and Optionally, the microdroplets can be subdivided and isolated for later recovery from the instrument. 3 zones; and It is configured to detect optical signals from merged microdroplets through one or more detection windows. This optical detection system detects the signal from merged microdroplets, and the reporter system also detects the cell. This is an optical detection system that arises from the interaction between its expression product and other related products.

2. The optical detection system includes a bright-field microscope, a dark-field microscope, means for detecting chemiluminescence, and Means for detecting Welster resonance energy transfer, and means for detecting fluorescence A device selected from the devices described in claim 1.

3. Is it integrated with the device, or is it a sorting component? A microlibrium is placed separately before or after, while any cells contained within are being cultured. Claim 1 or 2 further comprises a cell culture component configured to hold a droplet. The devices listed.

4. Cell culture components use optically mediated electrowetting forces The claim further configures to agitate the content of each microdroplet. The device described in 3.

5. It is configured to control the temperature of microdroplets in the cell culture component to 25-40°C. The device according to any one of claims 3 or 4 further includes a heater and a temperature controller. A chair.

6. We will generate an emulsion of microdroplets from biological samples in an immiscible carrier fluid. It is configured to be integrated with the device, or sorting Further including the aforementioned sample preparation component, which is placed separately before the component. The device according to any of the claims.

7. Sorting components, cell culture components, and sample preparation components At least one of the nets is operated in and / or between the droplets. A claim that is configured to have an electrowetting electrode position that enables A device as described in any one of items 3 to 6.

8. The sample preparation component has an electrowetting stretching force, and the biological sample preparation component has an electrowetting stretching force. The pull includes at least one position to which the pull is applied, from the biological sample to the carrier The device according to claim 6, configured to cleave microdroplets in a fluid.

9. The optical detection means is configured to collide with the first minute droplet in one or more detection windows. A detection system comprising a combined first electromagnetic radiation source for detecting fluorescence emitted from the minute droplets. The device according to claim 1, further comprising a container.

10. The microdroplet manipulation component includes one or more OEWOD structures, which include the following: The device according to any one of the claims: The first composite wall includes the following: First substrate A first transparent conductor layer on a substrate, the first transparent conductor having a thickness in the range of 70 to 250 nm. layer; The photoactive layer was activated by electromagnetic radiation with wavelengths of 400 to 850 nm on the conductive layer. The photoactive layer has a thickness in the range of 300 to 1500 nm. and A first dielectric layer on a photoactive layer, having a thickness in the range of 30 to 160 nm, layer; The second composite wall includes the following: Second substrate; The second conductive layer on the substrate, having a thickness in the range of 70 to 250 nm, body layer and Optionally, a second dielectric layer on a second conductor layer having a thickness in the range of 30 to 160 nm The second dielectric layer The exposed surfaces of the first and second dielectric layers are spaced 20 to 180 μm apart. , a second composite wall defining a microfluidic space adapted to contain minute droplets; A voltage is provided between the ends of the first and second composite walls that connect the first and second conductive layers. , A / C source; To induce the corresponding virtual electrowetting position on the surface of the first dielectric layer Higher energy than the band gap of the photoactive layer, adapted to collide with the photoactive layer. - at least one electromagnetic radiation source having; and The arrangement of virtual electrowetting positions is changed, thereby moving the microdroplets. To generate at least one electrowetting path that can be opened, light A means for manipulating the collision points of electromagnetic radiation on the active layer.

11. The first zone comprises an array of first microdroplet holding portions and a second zone including the reporter system. The first zone includes a reservoir with a port for introducing microdroplets, and the first zone is first The second microdroplet crosses the first microdroplet holding area so that the second microdroplet and the second microdroplet merge. The device according to claim 1, further configured to drive a droplet.

12. The device, via one or more paths of the virtual electrowetting electrode, The claim is that the second microdroplet is driven across the first microdroplet holding portion. The device described in 11.

13. The device receives a continuous stream of water in the first region of the device connected to the port. The second region of the device is configured to introduce microdroplets and overlaps the first region. However, in order to perform further operations, a microdroplet is guided from the first region to the third region of the device. The device according to claim 11, configured as follows:

14. An antifouling coating is provided on at least the surface of the first dielectric layer. The device described in item 10.

15. The performance of the sorting component, the first minute droplets into the first zone The introduction speed, and the first and second minute signals in response to the signals supplied by the detection system. A feedback loop controls one or more of the merging rates of small droplets. The preceding claim further comprises a microprocessor adapted to the above, as described in any one of the prior claims. The device.

16. The device is configured to illuminate and detect signals that characterize cells within the device. The device according to any one of the preceding claims, further comprising the optical assembly.

17. To manipulate and / or determine one or more characteristics of a cell type in a biological sample, A method for using the device described in any of the preceding claims, comprising the following steps Hmm, method: By creating aqueous first microdroplets in an immiscible carrier fluid from biological samples To create, and to create, which is believed to contain at least some cells of a specific cell type. thing; The first microdroplet is guided along the path using a real or virtual electrowetting electrode. to move to at least one microdroplet merging position; Path to the microdroplet fusion site using real or virtual electrowetting electrodes The characteristics of the cell type whose properties have been investigated in accordance with the aqueous second micro-dilution solution containing a reporter system characteristic of that cell type. Moving small droplets; Combining the first and second microdroplets at the merging position to produce a merged microdroplet; Beauty The content of each merged microdroplet is analyzed using an optical detection system, and the relationship between the cell and the reporter system is analyzed. To detect optical signals characteristic of the interaction between them.

18. The step of creating the first microdroplet contains both cell-containing microdroplets and empty microdroplets. Claim 17 further comprises the step of separating the cell-containing first microdroplets from a group of microdroplets containing other microdroplets. Methods used.

19. The first step of creating the microdroplets is performed under conditions that induce cell proliferation and division. The method according to claim 17 or 18, further comprising the step of culturing a group of small droplets.

20. The cell-containing first microdroplet is measured by an optical detection system, and the output of the system indicates that the state of the cell is measured. The method according to any one of claims 17 to 19, which sorts the results accordingly.

21. The first step of creating a microdroplet is performed by applying electrowetting stretching force. Claims 17-20 further include the step of cutting microdroplets from the biological sample. The method described in any one of the items.

22. The minute droplets are cleaved by the immiscible carrier fluid, and the immiscible carrier fluid The method according to claim 21, comprising a hydrocarbon or silicone oil.

23. An immiscible carrier fluid is optionally hydrated with aqueous micelles or secondary microdroplets of fluorocarbon. The method according to any one of claims 17 to 21, wherein the oil is carbon oil.

24. Culturing a group of microdroplets in an immiscible carrier fluid can lead to the formation of cells. This includes contact with a flow of a carrier fluid containing culture nutrients and / or dissolved gases. The method according to claim 19.

25. The dissolved gas comprises one or more of oxygen, nitrogen, and carbon dioxide, as described in claim 24. method.

26. The immiscible carrier fluid is periodically purged of gases harmful to the cell culture. The method according to claim 24.

27. Culturing a group of microdroplets creates a virtual electrowetting environment in which the microdroplets are retained. By applying an optically mediated electrowetting force at the electrode position, This includes stirring or agitating small droplets. The method according to any one of claims 19 to 26.

28. The reporter system responds to an enzyme or antibody expressed by cells of the desired cell type. and a selective reporter gene, cell surface biomarker or reporter molecule, The method according to any one of claims 17 to 27.

29. The reporter system detects the presence of enzymes or antibodies expressed by cells of the desired cell type. A luminescent reporter cell that selectively responds to the present, as described in any one of claims 17 to 27. Method of loading.

30. The optical detection system includes a bright-field microscope, a dark-field microscope, means for detecting chemiluminescence, and Means for detecting Welster resonance energy transfer, or means for detecting fluorescence One of the methods described in any one of claims 17 to 27.

31. The tiny droplets use electromagnetic waves to generate pathways for virtual electrowetting electrodes. Using an OEWOD structure adapted to the device, it is transported between positions on the device, and optionally, Claim 17 provides a stain-resistant coating and / or a biocompatible coating. The method described in any one of items ~30.

32. Using the device according to any one of claims 1 to 16, cell type in a biological sample A method for manipulating and / or determining one or more properties of a, comprising the following steps: By creating aqueous first microdroplets in an immiscible carrier fluid from biological samples To create, and to create, which is believed to contain at least some cells of a specific cell type. thing; The first microdroplet is guided along the path using a real or virtual electrowetting electrode. This involves moving to at least one microdroplet inspection position; and The content of each microdroplet is analyzed using an optical detection system, and the amount of cells contained in each microdroplet is determined to be one-third of the total cells. The above characteristics are determined, and one or more of the above characteristics are: cell morphology, cell movement, or at least one of the cell membrane integrity characteristics, which can be determined by analysis. and.

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