Device and method for microdroplet detection of cells

Abstract

To provide devices, systems and associated methods for manipulating and / or determining one or more characteristics of cells contained in a biological sample.SOLUTION: A device comprises: 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 electrowetting electrodes; and an optical detection system configured to detect an optical signal from the microdroplets via one or more detection windows.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] In accordance with the present invention, a device for rapid identification, manipulation and selection of cells is provided. and related methods are provided, which can be used to generate antibodies from immortalized cell culture samples or tissue samples. It is particularly useful for the manipulation of mammalian cells, either directly from a sample. It is also particularly useful for rapid, parallel screening of patient samples that may contain evidence of infection. It is useful. [Background technology]

[0002] Devices for manipulating droplets or magnetic beads have been previously described in the art. For example, US6565727, US20130233425 and US20150 For droplets, this result is typically achieved, for example, by mixing immiscible carriers. In the presence of a rear fluid, the droplets are deposited on two opposing walls of a cartridge or microfluidic tube. This can be achieved by moving the fluid through a microfluidic space defined by these Microelectrodes embedded in one or both walls of the device are covered with a dielectric layer, and each dielectric The electric layer can be rapidly turned on and off at intervals to modify the electric field properties of the layer. connected to an A / C bias circuit that steers the droplets along one or more predetermined paths. This produces localized, directional capillary forces in the vicinity of the microelectrode that can be used to measure the flow of fluid. Hereinafter, in connection with the present invention, we will use what we call "real" electrowetting electrodes. Such devices are called EWOD (Electrowetting on Diele This device is known in the art by the tric device.

[0003] The electrowetting force is optically mediated and is known in the art as optoelectronics. This variation of the approach is known as wetting and will be referred to below with the corresponding abbreviation OEWOD. Examples of formats include US20030224528, US20150298125, and US201 60158748, US20160160259, and Applied Physic s Letters 93 221110 (2008). In particular, these The first of three patent applications by The first wall is a composite design, including a substrate, a photoconductive layer and an insulating (dielectric) In this single-sided embodiment, various microfluidic devices are disclosed, each of which is comprised of a photoconductive The photoactive layer is electrically isolated from the insulating layer and is bonded to the photoactive layer. An array of conductive cells on which corresponding electrowetting electrode locations are generated At these locations, the surface tension characteristics of the droplets are similar to those of the electrowet droplets described above. The conductive cell then strikes the photoconductive layer. This approach may be useful for electrode placement. Although still somewhat limited by Furthermore, the speed at which the droplet can be moved and the actual droplet diameter can be controlled. There are limitations as to the extent to which the path can be changed.

[0004] A two-sided embodiment of this latter approach is presented by Pei at the University of California at Berkeley paper UCB / EECS-2015-119 In one example, a light-emitting diode (LED) on electrically biased amorphous silicon is The surface of the Teflon AF film was deposited on the dielectric layer using a pattern. Using electrowetting, relatively large droplets in the particle size range of 100–500 μm were obtained. However, in the illustrated device, the dielectric The layer is thin (100 nm) and is located only on the photoactive layer-bearing wall.

[0005] Our published application WO2018 / 010924, which is incorporated herein by reference in its entirety. In 234445, we use optoelectrowetting to provide the driving force. A device for manipulating microdroplets using optically mediated In electrowetting (OEWOD) devices, microdroplets contain walls. A microfluidic space defined by the microfluidic space, for example, a pair of parallel microfluidic spaces sandwiching the microfluidic space At least one of the containment walls is moved through the plate. This is referred to below as a "virtual" electrowet Selective illumination of layers with light from separate light sources. By this, it is possible to temporarily generate a virtual path of virtual electrowetting electrode positions. The microdroplets can be moved along it.

[0006] Our corresponding published patent WO2018, which is incorporated herein by reference in its entirety. / 234448 describes the use of this device as a working part of a nucleic acid sequencer. It is being done. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have developed a method for rapid screening and manipulation of biological samples containing cells. We used a microdroplet method similar to that underlying our previously described sequencer. A device for this application was developed. [Means for solving the problem]

[0008] Thus, in accordance with the present invention, one or more specific methods for analyzing cells contained within a biological sample are provided. 1. A device for manipulating and / or determining gender, the device providing: To be: configured to separate the cell-containing microdroplets from the empty space into a population of cell-containing first microdroplets. Sorting component created; First, we will demonstrate how to manipulate microdroplets using real or virtual electrowetting electrodes. A micro-droplet manipulation component configured as follows: Components: The first microdroplets are arranged in an array for optical inspection, and optionally, each microdroplet is merged to form a 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 device. 3 zones; and configured to detect an optical signal from the merged microdroplets via one or more detection windows. and an optical detection system, wherein the signal is transmitted from the reporter system to the cells or is an optical detection system resulting from the interaction between its expression product.

[0009] According to another aspect of the invention, a device according to the invention can be used to detect cells in a biological sample. A method for manipulating and / or determining one or more properties of a cell type, comprising the steps of: The law provides: by creating aqueous first microdroplets from a biological sample in an immiscible carrier fluid; at least some of which are believed to contain cells of a particular cell type. thing; The first microdroplet is then guided along a path using real or virtual electrowetting electrodes. and moving the microdroplet to at least one microdroplet inspection location; and The content of each microdroplet is analyzed by an optical detection system to determine the number of cells contained in that microdroplet. determining the above characteristics, wherein said one or more characteristics are selected from the following: cell morphology, cell movement, or or cell membrane integrity. and.

[0010] According to another aspect of the invention, a device according to the invention is used to detect a marker in a biological sample. 1. A method for manipulating and / or determining one or more characteristics of a cell type, comprising the steps of: Methods are provided: by creating aqueous first microdroplets from a biological sample in an immiscible carrier fluid; creating a population of cells, at least some of which are believed to contain cells of the cell type; and; First, the microdroplet is placed at least along the path using virtual electrowetting electrodes. Moving one microdroplet to a merging position; The virtual electrowetting electrode is used to measure the droplet size along the path to the droplet fusion point. Transfer a second aqueous microdroplet containing a reporter system characteristic of the cell type whose properties are being investigated. To make; merging the first and second microdroplets at a merging location to produce a merged microdroplet; and Beauty The content of each merged microdroplet was analyzed by an optical detection system to identify the relationship between the cell and the reporter system. and detecting optical signals characteristic of the interaction.

[0011] The 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) Cell-containing droplets from a population of microdroplets containing both cell-containing and empty droplets. separating the first microdroplet; (b) under conditions that cause cell growth and division before or after initial step (a) has taken place. culturing populations of microdroplets; (c) Electrowetting: Microdroplets are formed from biological samples by application of a stretching force. Generating a population of microdroplets by cutting.

[0012] In some embodiments of initial step (c), the microdroplets are formed from an immiscible carrier comprising an oil. The fluid contains the cut growth medium components, thereby allowing the initial step (b) to continue. Alternatively, the microdroplets may be filled with air or another gas mixture, creating an emulsion that can be cultured. For example, they are cut into a mixture of carbon dioxide and nitrogen and then cultured separately from each other. In some embodiments, the carrier fluid is periodically purged of gases harmful to cell culture. It may be advantageous to

[0013] In one embodiment of initial step (b), the culturing of the population of microdroplets is carried out in an emulsion and and a flow stream of an immiscible carrier fluid such as a hydrocarbon or a fluorinated oil, especially a fluorinated oil. This oil is used in the presence of surfactants and other additives to maintain the stability of the microdroplets. The oil may also be used to maintain cell growth during the culturing stage. The application of this embodiment involves the low levels of nutrients and gases required to sustain the The nutrient content of the microdroplets is periodically or continuously replenished by interfacial diffusion. Alternatively, nutrients and gases may be replenished 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, certain dissolved gases are purged from the oil to provide a low-oxygen environment for the cells. do.

[0014] In one embodiment of initial step (b), we use microdroplets to support cell growth. We have found that it is necessary to maintain optimal levels of certain atmospheric gases in the atmosphere. For example, this can cause deleterious changes in the pH of the droplet medium. In one embodiment, in initial step (b), the stream of immiscible carrier fluid is one or more saturated It contains a combined level of nitrogen, oxygen, or especially carbon dioxide.

[0015] In another embodiment of initial step (b), the content of the microdroplets is determined by the amount of the microdroplets retained. By applying an electrowetting force at the is agitated. Suitably, this is for example by agitation of the type described below. Optically mediated electrowetting force delivered by the OEWOD structure This is achieved using the method described above. The inventors believe that 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 for stirring or otherwise agitating a mixture is provided, the method comprising the steps of: Positioning the microdroplet at the virtual electrowetting electrode location; and Applying an electromagnetic radiation source to the location, thereby creating a corresponding virtual electrowetting The electrodes are activated and the electromagnetic radiation source is moved around the location, causing a corresponding movement and and the corresponding stirring or agitation of its contents. generating an associated electrowetting force, characterized by causing

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

[0017] For example, the device can be used to perform the following steps on male gamete cells, such as human or animal sperm cells: Inspection, selection and assay steps can be performed. In one example procedure, sperm cells The vesicle samples are prepared from diluted semen and encapsulated in droplets. The gamete-free droplets are then loaded onto the plate and examined using a bright-field microscope. Discard and retain any contained sperm cells for examination. Once a gamete is selected for analysis, a video of the gamete is taken along with a still image. The pattern recognition algorithms applied to the output from the This allows for the characterization of gametes in the droplets. The droplets can then be removed for further processing. , including on-chip assay steps such as addition of reporter reagents.

[0018] In another example, by encapsulating female gametes, such as human or animal eggs. It is possible to fertilize eggs. Similar to male gametes, female gametes are encapsulated in droplets. Once on the device, cells can be loaded onto the chip. can be examined for fluorologic defects and assayed using reporter reagents After inspection or assay, the female gamete cells may be subjected to any processing steps (e.g., via droplet motility). of embryonic epithelial cells by mechanical shear applied or by the addition of further reagents. removal).

[0019] In yet another example, male and female gametes are loaded onto a single microfluidic device. By merging droplets containing two gametes together and combining them In one example, multiple male gamete droplets merge with a single egg; Conventional interactions lead to fertilization and generation of blastocysts on-chip. The selected male gametes and the single selected and processed female gametes are combined on-chip. and interact with each other.

[0020] In another application, gametes of both sexes can be collected from a microfluidic chip and then administered via ICSI or IV. The mixture is combined using conventional handling techniques known in the art, such as F.

[0021] In some embodiments, the method may be performed by methods detailed above or known in the art. Blastocysts, which can be formed by conventional means, can be encapsulated in droplets and cultured on-chip. On-chip cultures can also be used to visualize the formation of cells using the imaging and detection systems described below. Using the droplet merging procedure, the blastocyst environment is adjusted to a buffer solution, salt , by adding extra materials such as nutrients, proteins, and extracellular matrix materials During blastocyst formation, a sample of cells can be removed from the blastocyst and stored for further analysis. Techniques such as laser microdissection are often used to retrieve them for further analysis. In some embodiments, the blastocysts are transported to a droplet manipulation zone. The pillars are then physically constrained between the electrowetting plates or the PCT / Electrowetting plates as described in EP2019 / 062791 This can include physical features on the microfluidic chip, such as a wedge-shaped change in the gap between the The disclosure of which is incorporated herein by reference. Once the blastocysts are loaded into the manipulation zone, Once attached, it is effectively fixed. Laser microdissection can then proceed. The process is well documented for removing part of the blastocyst. Once a portion of the droplet is excised, the droplet splitting procedure as described herein can be used to separate the scutellum. This can be used to separate a portion of the sample from the droplet. Distribution of material between the two droplets after splitting By repeated division and recombination procedures and machine vision inspection, the blastocyst and specimen parts are After separation, it is possible to verify that the blastocysts have been properly separated. Further analysis, such as by genetic testing including polymerase chain reaction or DNA sequencing, is needed. The product can be collected for analysis.

[0022] Suitably, the electromagnetic radiation source used in this method is a second electromagnetic radiation source as described below. and includes a rapidly flashing rotating light source that depicts a circular path in or around the location. In another embodiment, the movement of the light source may include one or more paths of lateral motion. In one embodiment, the position is defined by an area at least 0.5 microns in diameter, and the motion The motion can be circular, radial, or a mixture of the two. radial to produce responsive centrifugal mixing.

[0023] In principle, the reporter system that can be introduced into the first microdroplet in step (4) can be any of the following: characteristic for assaying the presence of a given cell type or cell behavior in a biological sample or any system that can be used. Such reporter systems include, for example, , against an enzyme, protein, or antibody expressed by cells of the desired cell type. Selective reporter genes, cell surface biomarkers or reporter molecules A related class of reporter systems involves the detection of relevant material expressed by the cells being probed. The second reporter cell may be a cell that responds to the presence of a specific gene. Many such assays are known, and suitable candidates for use will often be apparent to one skilled in the art. A different reporter system is introduced into the first microdroplet by merging with one or more second microdroplet types. By incorporating a range of different cells, the method allows for the identification of a range of different properties and behaviors. It will be appreciated that the method may be multiplexed to perform parallel and simultaneous searches for rule types. can be. [Brief explanation of the drawings]

[0024] [Figure 1] Referring to Figure 1, an example device and associated workflow are shown. [Figure 2] Figure 2 shows a cross-sectional view of an exemplary device, an oEWOD structure with a diameter of 120 μm (e.g., 80-120 μm) in an unconfined state, suitable for rapid manipulation of aqueous microdroplets 2 emulsified in a fluorocarbon oil with a viscosity of 5 centistokes or less at 25 °C. DETAILED DESCRIPTION OF THE INVENTION

[0025] The method of the present invention and its various steps and initial substeps are described below. The method can be conveniently carried out using an analytical device of the type described above. Examples of are also described below. In some embodiments, the device includes: A sorter for separating cell-containing microdroplets into a population of cell-containing primary microdroplets from empty ones. ting components; Subsequent manipulation of the first microdroplets using real or virtual electrowetting electrodes 1. A micro-droplet manipulation component for performing micro-droplet manipulation, comprising: nt: means for introducing a reporter system into each first microdroplet by microdroplet merging; Zone 1; The merged droplets are then detected in one or more detection windows in or near the first zone. Zone 2, located adjacent to and The reporter system can be used in combination with bright-field microscopes, dark-field microscopes, chemiluminescent detection means, Förster microscopes, and The interaction with the cell or its expression product selected from the resonance energy transfer detection means or the fluorescence detection means. An optical detection system that detects optical signals from the merged microdroplets resulting from the interaction.

[0026] The sorting component used in such an embodiment may comprise one or more cells. The microdroplets (hereafter referred to as "filled microdroplets") are then separated into a larger population of droplets, some of which are empty. Depending on the type of sorting component selected, Therefore, the microdroplets can be classified into two different microdroplets depending on whether they are filled or empty. Directed towards or down one of the fluid paths or receiving locations. In embodiments, access to one or the other of these allows for analysis of each microdroplet within the analysis window. The system is controlled by the gate or actuation of an electromechanical gate that acts in response to the analysis. In this form, sorting is performed by a responsive optically mediated electrowetting technique within the analytical window. A flicking force is applied to the stream of microdroplets to pull selected ones into a holding area or array. This is achieved by allowing the rejected microdroplets to remain in the airflow. In another embodiment, the sorting decision is based on an optical phenomenon. based on bright field microscopy, or by optical properties associated with the cells, e.g., fluorescent tags or In another embodiment, sorting is by detecting the presence of a transient marker. A suitable electric field is applied to the analysis window to force each droplet into one of two different paths on either side of the partition. In one embodiment, this can be achieved by dielectrophoresis, which deflects the liquid toward the surface. The sorting component comprises a first microfluidic channel terminating in an analysis chamber and a second microfluidic channel terminating in an analysis chamber. At least two first microdroplets are connected to a downstream analysis chamber, at least one of which carries away the first microdroplets. 2 microfluidic channels, a light source for illuminating the analysis chamber, and a light source for illuminating each illuminated microfluidic channel within the analysis chamber. Brightfield microscope or fluorescence detector for acquiring data from droplets and two second channels at least one OEWOD structure operable to direct a microdroplet to one of the Depending on the results from a discrimination algorithm applied to the data received from the mirror or fluorescence analyzer and a microprocessor adapted to operate the structure in response.

[0027] In one embodiment, the device may include an integral component of the device itself. or before or after the sorting component, preferably The method further comprises a culture component separately disposed after the feeding component, wherein: The microdroplets are retained, but any cells contained within are stimulated to divide and grow. Preferably, the culture component is configured so that the microdroplets are cultured under optimal culture conditions. Typically, a container that is kept at a temperature above 25°C (e.g., 25-40°C) for 1 hour to 1 week and an inlet for introducing microdroplets therein. The device includes a thermostatically controlled heater and, optionally, a and a timer to control the cycle. In one embodiment, the device contains The device comprises an emulsion of microdroplets in a compatible carrier fluid, and the carrier fluid changes over time. The device further includes an inlet and an outlet through which the device can be replaced. In one embodiment, the immiscible carrier fluid is HFE7500, HFE7700, or FC Fluorocarbon oils such as -40. Such oils maintain the stability of the microdroplets. and to maintain low levels of nutrients and gases necessary to sustain growth, It may further suitably contain anti-oxidants and other additives.

[0028] In one particularly useful embodiment, where the weight ratio of aqueous to oil microdroplets is low, the microdroplets are dispersed over time. Both tend to shrink, which can lead to a loss of reactivity within the microdroplets. One way to counteract this effect is to use hydrated oils. Since oil does not have a high capacity to dissolve water, hydration occurs when water or aqueous buffer is added to the oil phase. This is suitably achieved by forming micelles or secondary microdroplets of the solution. The composition of the microdroplets may be the same as or different from that of the microdroplets themselves. In this state, these micelles or secondary microdroplets contain up to five times the salt content of the microdroplets themselves. These micelles and dimers may optionally contain glycerin. The next microdroplets are an order of magnitude smaller.

[0029] In one embodiment, the container is and a plurality of locations where microdroplets can be placed and agitated to agitate their contents. The surface further includes a surface that can be

[0030] The device may further comprise a device that is integral to the device itself or that receives an aliquot of a biological sample. a means for producing an emulsion of microdroplets in an immiscible carrier fluid from the culture medium; The sample preparation component may be either located separately or upstream of the nutrition component. nothing.

[0031] This sample preparation component involves cleaving microdroplets from a biological sample into a carrier fluid. the cutting means includes a cutting means for cutting the biological sample by applying an electrowetting elongation force to the biological sample. In one embodiment, the cutting means comprises: a first electrowetting location adapted to receive a biological sample; The first and second electrowetting electrode positions are directional electrowetting to define a path along which the microdroplets cleaved from the sample can be transported using a force. at least one second electrowetting location disposed thereon; a first electrowetting position and an electrode and associated AC current AC, consisting of either a circuit or a semiconductor zone activated by the impingement of electromagnetic light Drive circuit and A first electrowetting position is positioned to electrostatically charge the surface of the biological sample. A DC charging circuit adapted to charge a

[0032] In one embodiment, the disconnecting means is connected to the drive circuit and the charging circuit, which are suitably AC and DC circuits. In another embodiment, the disconnection means generates a control circuit for switching between the power supply and the power supply. It further includes an analyzer for analyzing the content of each microdroplet generated from the biological sample. In this regard, a biological sample may be blood, plasma, sputum, urine, or a sample derived from a tissue biopsy. The cleavage may be performed in any aqueous medium, such as a tissue. This can be found in our co-pending application EP18201162.7, to which the reader is directed. The microdroplet cutting method associated with the cutting means forms the basis for carrying out the first step (c) above. It will be readily apparent that this can be achieved.

[0033] Used to later manipulate the first microdroplets generated by the sorting component. Turning to the microdroplet manipulation component used, this suitably comprises a first zone and The second zone and the first microdroplets are attracted by aerodynamic forces and / or electrowetting forces. the actual fluidic devices connected to one another by one or more microfluidic pathways that are driven along the pathways. and an optical detection system consisting of real or virtual electrowetting electrodes. Preferably, the electrowetting electrodes are virtual and are located on one or more Generally, this is done by manipulating the microdroplets in the present method. In one embodiment, these OEWOD structures include: The first composite wall contains: First board A first transparent conductor layer on a substrate, the first transparent conductor layer having a thickness in the range of 70 to 250 nm. ; On the conductive layer was a photoactive layer activated by electromagnetic radiation with wavelengths of 400 to 850 nm. and a photoactive layer having a thickness in the range of 300 to 1500 nm. and a first dielectric layer on the photoactive layer, the first dielectric layer having a thickness in the range of 30 to 160 nm; ; Secondary composite wall containing: Second board; a second conductor layer on the substrate, the second conductor layer having a thickness in the range of 70 to 250 nm; body layer and Optionally, a second dielectric layer on the second conductor layer, having a thickness in the range of 30 to 160 nm. a second dielectric layer wherein the exposed surfaces of the first and second dielectric layers are spaced apart by 20 to 180 μm. a second composite wall defining a microfluidic space adapted to contain a microdroplet; providing a voltage across the first and second composite walls connecting the first and second conductor layers; , A / C source; to induce corresponding virtual electrowetting positions on the surface of the first dielectric layer. the photoactive layer is adapted to impinge on the photoactive layer at an energy higher than the band gap of the photoactive layer. at least one electromagnetic radiation source having a The configuration of the virtual electrowetting location is changed, thereby displacing the microdroplets. to generate at least one electrowetting path that can A means for manipulating the impingement point of electromagnetic radiation on the active layer.

[0034] In one embodiment, the first and second walls of these structures sandwich a microfluidic space. Alternatively, the first substrate and the first conductor layer may be connected to a source of electromagnetic radiation (e.g., a multi-laser to allow light from a photoactive layer (e.g., a photobeam, lamp, or LED) to impinge on the photoactive layer. Alternatively, the second substrate, the second conductor layer, and the second dielectric layer may be transparent. In yet another embodiment, the layers are all transparent.

[0035] Suitably, the first and second substrates are made of a mechanically strong material, such as glass, metal or alloy. In one embodiment, the substrate is made from a flexible plastic. In yet another embodiment, the first and second substrates may have a molecular weight of 100 to 1 In some embodiments, the first substrate has a thickness in the range of 100000 μm. In some embodiments, the second The substrate is composed of one of quartz glass and glass.

[0036] The first and second conductor layers are located on one surface of the first and second substrates, and are typically has a thickness in the range of 70 to 250 nm, preferably 70 to 150 nm. In this case, at least one of these layers is made of a transparent material such as indium tin oxide (ITO). From very thin films of conductive materials, such as silver, or conductive polymers such as PEDOT These layers are formed as a series of discrete structures, such as a continuous sheet or wire. Alternatively, the conductor layer may be made of a conductive material that allows electromagnetic radiation to be directed between the gaps in the mesh. It may also be a mesh of material.

[0037] The photoactive layer is capable of generating localized regions of charge in response to stimulation by a second electromagnetic radiation source. Suitable semiconductor materials are available. Examples include those with thicknesses in the range of 300-1500 nm. In one embodiment, the photoactive layer may include a hydrogenated amorphous silicon layer having a thickness of 1000 Å. 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 to 500 nm thick. The substrate is coated with a dielectric layer with a thickness in the range of 160 nm. The dielectric properties of this layer are > It preferably includes a high dielectric strength of 10^7 V / m and a dielectric constant of >3. It is as thin as possible consistent with avoiding dielectric breakdown. 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 layers, preferably both, are Desired microdroplets / carrier fluid / surface at various virtual electrowetting electrode positions It helps establish a contact angle and also helps the contents of the microdroplet to adhere to the surface and To prevent droplets from being lost as they move through the tip, the chip is coated with an anti-fouling layer. If the second wall does not include a second dielectric layer, the second antifouling layer is For optimum performance, the anti-smudge layer should be applied to an air-liquid-surface Microdroplet / carrier fluid / surface contact angles of 50 to 170° when measured as a three-point interface In one embodiment, these layers have a thickness of less than 10 nm. In another embodiment, these layers have hydrophilic groups, e.g. For example, alkoxysilyl-substituted acrylic esters, such as methyl methacrylate or One or both of the anti-fouling layers may be coated with a polymer of the derivative thereof to ensure optimum performance. In some embodiments, the 20 By interposing a silica interstitial layer of less than 100 nm thickness between the antifouling coating and the dielectric layer, This can be done.

[0040] The first and second dielectric layers, and therefore the first and second walls, have a width of at least 10 μm. microfluidic cavity, preferably in the range of 20 to 180 μm, in which the microdroplets are contained; Preferably, before they are contained, the microdroplets themselves define a width of the microdroplet space. By this means, the characteristic diameter of the Upon entering the cap, the microdroplets are compressed, e.g., through better microdroplet merging ability. This improves the electrowetting performance.

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

[0042] In another embodiment, the microfluidic space maintains the first wall and the second wall separated by a predetermined amount. The spacer includes one or more spacers to support the optical pattern. Beads, pillars, and ridges formed from the intermediate resist layer produced by etching Alternatively, deposition materials such as silicon oxide or silicon nitride can be used to form the surface. Alternatively, a flexible plastic with or without an adhesive coating may be used. Layers of film, including plastic film, can be used to form the spacer layer. Various spacer shapes are used to create a narrow tapered channel defined by the pillar lines. With careful design, open or partially enclosed channels can be formed. Thus, these spacers can be used to aid in the deformation of the microdroplets, and subsequently Droplet splitting and effect manipulation on the deformed droplets can be performed. These spacers are used to physically separate zones of the chip and prevent cross-contamination between droplet populations. Prevents contamination and promotes well-directed droplet flow when loading the tip 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. , providing a voltage potential difference between them, suitably in the range of 10 to 50 volts.

[0044] These OEWOD structures typically have a wavelength of 400 to 850 nm, preferably 660 nm. a second electromagnetic wave having a wavelength in the range of 1000 .ANG. and an energy 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 intensity of the radiation is 0. 0.1~0.2 Wcm -2is activated at a virtual electrowetting electrode position The electromagnetic radiation source has a corresponding photoexcitation region on the photoactive layer, which in one embodiment is also pixelated. By this means, the pixelated virtual electrowet A bonding electrode position is introduced onto the first dielectric layer.

[0045] If the electromagnetic radiation source is pixelated, it is illuminated by light from an LED or other lamp. Direct or using a reflective screen such as a digital micromirror device (DMD) A suitable source of electromagnetic radiation is provided, either directly or indirectly, to create a virtual electrowetting electrode location. Highly complex patterns can be rapidly created and destroyed on the first dielectric layer. This allows for the formation of microdroplets at essentially any size using tightly controlled electrowetting forces. This allows for precise steering along virtual paths, which is a technique that allows the chip to move along multiple paths. It is also particularly advantageous when thousands of such microdroplets need to be manipulated simultaneously. Such an electrowetting path is a virtual electrowetting path on the first dielectric layer. The sigma can be seen as being constructed from a continuum of moving electrode positions.

[0046] The point of impingement of the electromagnetic radiation source on the photoactive layer may be of any convenient shape, including conventional circular or annular shapes. In one embodiment, the morphology of these points can be expressed as a corresponding pixelated morphology. The shape of the microdroplets entering the microfluidic space is also determined by the In one embodiment, the impingement point, and therefore the electrowetting electrode location, corresponds to a crescent moon. The shape of the droplet may be oriented in the intended direction of travel of the droplet. The wetted electrode position itself is smaller than the microdroplet surface adhering to the first wall, and the droplet and the surface dielectric gives the maximum electric field strength gradient across the contact line formed between

[0047] In one embodiment of the OEWOD structure, the second wall is illuminated by the same or a different electromagnetic radiation source. The photoactive layer also allows for the induction of virtual electrowetting electrode positions on the dielectric layer of the second electrode. The addition of a second dielectric layer improves the transition of the wetted edge of the microdroplet from the top surface to the bottom surface of the structure. , and allows for the application of more electrowetting force to each microdroplet.

[0048] A first zone forming part of the device, in one embodiment, introduces a first microdroplet. an inlet for the electrode and an outlet attached to the second zone by an electrowetting path. The first zone is a port for introducing a reporter system. In one suitable embodiment, the reporter system further comprises A second aqueous microdroplet containing a reporter system designed to identify the properties of the contained cells. In one embodiment, the reservoir holds the first microdroplet. and further comprising an array of locations over which the second microdroplet is driven. , which process causes some merging of the first and second microdroplets. The merged first / second microdroplets (hereinafter referred to as "merged microdroplets") are those in which the reporter system They interact sufficiently with the cells so that they are then transported to the second It may be held in that position until it generates an optimal optical signal to be transported to the zone. In some cases, it may be desirable to monitor the growth of the optical signal using time-resolved measurements. In an embodiment, a single zone encompasses the roles of both the first and second zones, e.g., This is used by detecting the merged microdroplets at the merger location mentioned above.

[0049] The second zone preferably includes one or more detection windows through which the light can be detected using an optical detection system. In one embodiment, the second zone is a chip. In another embodiment, the optical detection system detects the reporter system and the cells or their expression. It is designed to detect optical signals from microdroplets resulting from interactions between the liquid and the product. Suitably, the optical detection system may be bright field, dark field, or chemiluminescence. means for detecting Förster resonance energy transfer or means for detecting fluorescence In one embodiment, the detection system is selected from means for detecting the merged microdroplets and / or receives a signal from one of the detectors and displays it, for example in the form of a visual display or counting It also includes a light source for illuminating the microprocessor for providing data to a user in a read-only state. In one embodiment, the microprocessor determines the performance of the sorting component, the first zone, The speed of the first microdroplet introduction into the nozzle and the signal detected by the optical detection system were measured. To control one or more of the coalescence rates of the first and reporter system-containing microdroplets, It is further adapted by a feedback loop.

[0050] A particular advantage of instruments using the oEWOD structure for performing droplet manipulations is the ability to focus the sample. The key feature is that the optical addressing system is built into the fixture. By multiplexing and demultiplexing the excitation and emission light required for light detection along with the necessary illumination, This allows much of the optical functionality to be combined into one simpler, lower-cost assembly. For example, a long-pass dichroic mirror can be used to capture the emitted light from the assembly. It is possible to multiplex it, divert light from the operation column, or turn it into a high-sensitivity detection camera. Another embodiment uses two dichroic mirrors. The first mirror directs the light from the lamp to the A second mirror is used to multiplex the fluorescence emission light. This requires more sophisticated illumination schemes such as time-resolved Förster resonance energy transfer. In the embodiment, for applying a time-dependent and spatially varying illumination pattern, It is preferable to employ the same structured illumination system to address the EWOD scanning pattern. , as well as using dichroic mirrors for these multiplexed operations, dispersion filters It is possible to use elements such as filters, dispersive lenses or diffraction gratings. This involves implementing temporal multiplexing, where a structured illumination system is used to rapidly switch between excitation sources. It is preferable to carry out

[0051] In some embodiments, the device ejects microdroplets from the first and / or second zones. and a third zone for collecting the first microdroplets, e.g., as described in more detail below. The fragments may be subdivided and isolated for later recovery from the device for subsequent or confirmatory analysis. In one embodiment, the third zone is connected to one or more of the second zones connected to the temporary storage container. consists of multiple egress ports. and sequentially collecting multiple droplets by refilling the second zone. , allowing multiple droplets to be collected separately from one port.

[0052] As well as optically mediated manipulation of fluids within the OEWOD structure, the device also The fluid flow inside the device is controlled by selectively applying fluid pressure to the inlet and outlet ports. It may include a network of pumps and valves for operating it. The network consists of a two-position valve connected to each outlet, and a pair of pressure sources (potential pressure sources) connected to the same valves. By changing the configuration of each valve, Positive or negative pressure can be applied within the device via the reservoir and collection vessel. , thus, the inflow of material into the device, the outflow of material out of the device, or the This causes an influx of material into the

[0053] The above-described inventive device and related methods have many beneficial applications. We will describe some example applications and the associated workflows.

[0054] One application of the disclosed devices and methods is in the development of genetically modified cell lines. be.

[0055] In this application, target cells are sampled using optically mediated enzymes in the sample preparation component. via electrowetting-based cutting means, The transfection reagent (e.g., modified lentivirus) is delivered to a separate, second microfluidic vessel. encapsulated in droplets.

[0056] The first and second microdroplets are then merged into the OEWOD device in a merging operation as described above. The droplets merge on the chair to form merged microdroplets, which are then used to transfect target cells. The cells in the merged microdroplets are exposed to a phospholipid reagent. The cell population is divided between the droplets. The cells are subjected to cycles of merging and splitting, and the medium surrounding the cells is diluted by serial dilution. Fresh medium is replaced to replenish depleted materials and any cellular waste that has accumulated within the droplet. Remove things.

[0057] For example, the position of each cell in the microdroplet population can be tracked by microscopy during the assay. Tracing allows cells derived from a common ancestor to be identified for sorting purposes. This ensures the monoclonality of the cultured cell population.

[0058] During the described process, freeze-thawing, which is known to reduce cell viability, Cell retention is also improved as there are no harmful steps of dispensing, manual handling, or repeated long-term passaging. Increased cell density compared to traditional cell-based development methods. Similarly, cells remain encapsulated within the droplets. This eliminates the possibility of losing clones to the liquid handling device surface. The cells are found early in the process and can be replaced by viable cells.

[0059] Once it is determined that the cells in the fused microdroplets have been cultured for a sufficient length of time, Upon detection, a third reagent containing a reporter assay is introduced into the OEWOD device, which binds to the target cells. The results of the reporter assay can be measured, for example, by detecting fluorescence, It can be measured according to chemiluminescence or Förster resonance energy transfer.

[0060] Based on the results of the reporter assay, one or more first subsets of cells may be discarded. The sample may then be cultured and one or more second subsets of cells may be further expanded. - Target cell subsets are collected and removed from the chip for further off-chip analysis The cultured subsets are distributed into well plates, e.g., standard 1536-well plates. The remaining progenitor cells from the patient are retained for further culture on-chip.

[0061] The recovered samples are then subjected to one or more off-chip analyses (e.g., DNA sequencing, RNA sequencing, A) sequencing, PCR analysis, gene profiling, and microarray measurements Based on the results of the off-chip analysis, a further subset of cells is selected for on-chip analysis. The cells can be cultured, harvested, and further cultured.

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

[0063] After immunization with an antigen, such as a toxin or biomarker characteristic of a disease, B cells, T cells, or Samples of natural immune cells, such as dendritic cells or dendritic cells, are collected from living organisms such as mice, humans, or primates. The cells can then be harvested from surrounding tissue, lymph, blood cells from the host organism. The cells are processed and purified to separate them from the cells and other components. This can involve dissection, centrifugation, This can be achieved by a combination of immunoprecipitation, filtration and dialysis methods.

[0064] Purified immune cells are encapsulated in microdroplets and loaded onto the OEWOD device Then, immunoassay reagents, FRET reporters, or reporter cell lines are used. Which reagents are introduced into the microdroplets containing the target cells in the first assay, This is performed by generating a second microdroplet of the reagent and performing a merging operation as described above. It is possible.

[0065] The results of the first assay are examined, for example, by optical detection or microscopy, and the antibody Proteins such as are excreted by target cells in response to immunoassay reagents. Based on the results of the first assay, a sample of the microdroplets containing the target cells is determined. A subset can be discarded from the device and another subset retained for further testing. will be done.

[0066] A second reagent, such as an off-target reporter, is then added to the second on-chip assay. The remaining cell-containing microdroplets may then be introduced in the same manner. The results are measured using optical techniques such as fluorescence spectroscopy and are compared with the results of a second round of microdroplet subcell analysis. The above process is performed using a series of different reporter assays. Interrogate / screen target cells for on-target, off-target, and unrelated targets The test can be repeated to measure the response of the target cells to the test.

[0067] As used herein, the term "on-target" refers to the tissue or antigen at the end. The target cells produce responsive antibodies. For example, the target may be cancerous tissue. As used herein, the term "off-target" refers to any undesired effect observed. Off-target refers to tissues that are close to or associated with cancerous tissue. As used herein, the term "irrelevant target" refers to a target that is a healthy tissue. is expected to have no biological interaction with the antibody, but may, for example, provide a large amount of useful terminal Substances that may have a negative effect on the accuracy of the assay results by binding to antibodies or by confounding measurements This refers to

[0068] The final subset of cells is selected based on the results of the screening assay. The remaining cell-containing microdroplets containing the final subset are then lysed with the selected lysis reagent and cD NA synthesis reagent to generate a library of genes currently expressed in the target cells. The cells of interest are collected off-chip and the behavior observed in the on-chip phenotypic assay is analyzed. The resulting DNA is then subjected to a genetic assay that reveals the coding DNA involved in the process.

[0069] Another application of the present invention is the functionalization of drugs, including immunomodulatory drugs and drugs for tumor suppression. and screening for efficacy.

[0070] In this application, a panel of drug target cells is encapsulated in a first microdroplet. and loaded onto the OEWOD device. A second set of microdroplets containing the same is also loaded.

[0071] The dosimetry panel is a panel of microdroplets containing a range of dilutions of each drug compound. The second microdroplet is formed by a merging and splitting operation performed on the second microdroplet to create the The drug compound may be encapsulated in vesicles, even in the form of microbeads. may also be expressed by a producer cell encapsulated in the droplet.

[0072] The drug dosimetry panel is introduced into the target cells by a merging operation; drug dosimetry Exhaustive pairwise combinatorial processes between the first cell and the microdroplets containing the panel The process ensures that the entire panel is exposed to every cell type in replicates. Master cells (e.g., T killer cells or macrophages) also play a role in regulating immune responses. to test the effects of drugs and perform detailed cross-sectional comparisons of immune responses in the presence of different tissues To achieve this, the antibody can be introduced with a panel of drugs and target cells.

[0073] The response of target cells to the drug panel can be assessed, for example, by microscopy, fluorescent reporter staining, or The results of the screening process can be used to monitor the to provide information about the efficacy of test drugs in various cellular 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 placed in an OEWOD device. loaded on top.

[0076] For example, growth factors, environmental stimuli, cell-to-cell signaling compounds, and molecules A panel of control reagent compounds, such as phosgenes, are encapsulated in the second microdroplet, and It is loaded onto the OEWOD device.

[0077] A first subset of microdroplets exposes the stem cells contained in the first microdroplets to a reagent, and Therefore, to promote differentiation of stem cells along the target pathway, a second microarray containing a control reagent is used. The droplets are merged.

[0078] The stem cell differentiation process can be visualized using, for example, microscopic imaging, detection of phenotypic reporter compounds, and labeling. The differentiated cells in the merged microdroplets are monitored by performing a transporter assay. Cells are recovered from the OEWOD device via an aliquoting step for further culture or processing. It can be done.

[0079] Yet 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 first and loaded onto an OEWOD device. Regulatory reagents such as growth factors, environmental stimuli, intercellular signaling compounds, and morphogens The panel is encapsulated in a second microdroplet and also loaded onto an OEWOD device. will be done.

[0081] The first microdroplet contains a subset of the organoid progenitor cell population, which is then transformed into organoids and In order to promote the formation of tissue structures, the cells are exposed to a control agent via the merging procedure. The organoids formed by this method are stored on-chip in a dedicated area on the OEWOD device. The cells can be supplied with nutrients and any other necessary growth medium via a droplet merging operation. Organoids stored on a chip in the manner described above in connection with the exemplary application. Thus, they can be used in drug screening assays.

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

[0083] In this application, target cells are encapsulated in the first microdroplet and then injected into the OEWO. A second set of microdroplets containing a panel of gRNA pairs is also loaded onto the device. The panel of gRNA pairs is then loaded onto the device. The gRNAs were spotted onto the target regions on the device surface, and then microdroplets were placed over them. The preparation step may include rehydrating the area containing the panel by passing it through a

[0084] This involves attaching gRNA to microbeads, spotting these beads, and then eluting them with fluid. This can be in the form of a bead prep process where the beads are freeze-dried onto the surface of the beads.

[0085] The gRNA is introduced into the target cells via the merging procedure, which allows the target cells to , programmable restriction enzymes (e.g., Cas9) and gene modification in target cells The gRNA is incorporated along with the reagents required to induce mutation.

[0086] The cells in the merged droplets are separated into two droplets, and the medium surrounding the cells is separated into two droplets. It is subjected to cycles of combining and dividing operations alternated by serial dilutions.

[0087] Sorting cells derived from a common ancestor by tracking the position of each cell in the microdroplet population This allows for the identification of cells for screening purposes, increasing the monoclonality of the cultured cell population. Holdings also increase.

[0088] Once fused, it is determined that the cells in the microdroplets have been cultured for a sufficient length of time. Then, a third reagent containing a reporter assay is introduced into the OEWOD device, and the target cells are The results of the reporter assay can be measured, for example, by the detected fluorescence, chemical It can be measured according to luminescence or Förster resonance energy transfer.

[0089] Based on the results of the reporter assay, one or more first subsets of cells may be discarded. The sample may then be cultured and one or more second subsets of cells may be further expanded. - Target cell subsets are collected and removed from the chip for further off-chip analysis The cultured subsets are distributed into well plates, e.g., standard 1536-well plates. The remaining progenitor cells from the patient are retained for further culture on-chip.

[0090] The recovered samples are then subjected to one or more off-chip analyses (e.g., DNA sequencing, RNA sequencing, A) sequencing, PCR analysis, gene profiling, and microarray measurements Based on the results of the off-chip analysis, a further subset of cells is selected for on-chip analysis. The cells can be cultured, harvested, and further cultured.

[0091] Referring to Figure 1, an example device and associated workflow are shown.

[0092] Fluid inlet 1 contains an emulsion of a mixture of empty cell-containing first microdroplets in fluorocarbon oil. These first microdroplets are then transferred to the OEWOD structure (not shown). and transferred to sorting zone 3, where they are sorted by optical means or other sorting methods as described above. By the filtering means, the cells are sorted into empty ones 4 and ones containing cells 5. Each of the contained microdroplets 5 is transferred, also by the OEWOD structure, to the confluence zone 8, where They are maintained for a defined period of time under conditions that promote cell growth and division within each. At the end of this period, a second inlet 6 is introduced into the cell via a fluorescent reporter selective for the cell type 7 of interest. These then merge with the cell-containing first microdroplets at the confluence zone 8. The droplets 5 are merged to form merged microdroplets 9. The merged microdroplets 9 are then The EWOD structure transfers the fluorescent signal characteristic of the reporter system to the optical window 10, where it is The detection is performed using an optical detection device 11 consisting of an LED light source, a photodetector, and a microprocessor. The optical detector 11 is partially combined with an optically scanned projector 12.

[0093] Figure 2 shows the viscosity of a fluorocarbon oil with a viscosity of less than 5 centistokes at 25°C. 120 μm in unconfined state (e.g., The cross-sectional view of an exemplary device is shown, which shows 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. The upper and lower glass plates (13 and 14) were coated to a thickness of 500 μm, respectively. ) Each of 15 is connected to an 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. 17 are each coated with a 160 nm thick layer of high purity alumina or hafnia 18. Then, trichloro(1H,1H,2H,2H-perfluorooctyl)silane 19 The silicon dioxide supporting layer is coated with an interstitial layer of silicon dioxide, resulting in a hydrophobic surface. 13 and 17 are designed so that the microdroplets undergo some compression when introduced into the device. The reflective image is illuminated by the LED light source 20 and spaced 80 μm apart using spacers. The image on the pixel screen is generally placed below 14 and has a brightness of 0.01Wcm -2 Visible light (wavelength 66 0 or 830 nm) is emitted from each diode 21 and passes through 14 and 15 It is caused to collide with 17 by propagating in the direction of the multiple upward arrows.

[0094] At various points in the collision, photoexcited regions of charge 22 are transferred to corresponding electrowetting At the contact position 23, a liquid-solid contact angle is generated at 17, which is modified at 18. These improved properties are necessary to propel the microdroplet 2 from one point 23 to another. 20 provides capillary force. The array is arranged at a time by a pre-programmed algorithm. The control is controlled by a microprocessor 24 which determines which of the four beams 21 is irradiated. .

Claims

1. 1. A method for screening for drug functionality and efficacy, comprising the steps of: encapsulating a panel of drug target cells in a first microdroplet; loading the first microdroplet onto an oEWOD device; introducing a second set of microdroplets containing a panel of drug compounds; introducing the second microdroplet into the first microdroplet by a merging operation in an exhaustive pairwise combination process; and Monitoring the response of the drug target cells to the drug compound to provide information about the efficacy of the drug compound.

2. 10. The method of claim 1, further comprising combining effector cells into said combined microdroplets.

3. The method of claim 2, wherein the effector cell is a T killer cell or a macrophage.

4. The method of any one of claims 1 to 3, wherein the monitoring step is carried out by microscopy, fluorescent reporter staining or reporter assay.

5. The method of claim 1, wherein the second set of microdroplets is formed by a merging and splitting operation to provide a range of dilutions of the drug compound.

6. 6. The method of any one of claims 1 to 5, wherein the drug compound is in the form of a microbead, encapsulated in a vesicle, or expressed by a producing cell encapsulated in the second droplet.

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