Microdroplet manipulation device

Abstract

To provide a device suitable for manipulation of an emulsion of microdroplets and carrier fluid.SOLUTION: A device for manipulating microdroplets comprises a microfluidic chip adapted to receive and manipulate microdroplets dispersed in carrier fluid flowing along pathways therethrough, where the chip includes regions of differing or zero microdroplet fluid flow rates. Also disclosed is electrowetting means of transporting a plurality of kinds of emulsions and components of emulsions between the different flow regions.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microfluidic chip suitable for manipulating emulsions of microdroplets and a carrier fluid. In the microfluidic chip, the components of the emulsion are selectively mixed with the emulsion along with a retention force that is selectively applied. Agents can be independently manipulated by exposing them to different flow regions.

[0002] Devices for manipulating droplets and magnetic beads have been previously described in the art. For example, see Patent Documents 1 to 3. In the case of droplets, this result is typically The droplets are then transferred to two opposing channels of a cartridge or microfluidic tube in the presence of a mismatched carrier fluid. This can be achieved by passing the liquid through a microfluidic space defined by walls that are spaced apart from each other. One or both walls of the ridge or microfluidic channel are embedded with microelectrodes covered with a dielectric layer. Each electrode is connected to an A / C bias circuit. can be rapidly turned on and off at intervals to change the electric field properties of the layer. This generates a localized directional capillary force near the microelectrode, which can be used to separate the droplets. Such a device can be used to guide the user along the above-mentioned predetermined route. The technology uses what are known in the industry as "true" electrowetting electrodes. is known by the acronym EWOD (Electrowetting on Dielectric) Device.

[0003] Variations on this approach in which the electrowetting force is optically mediated are known in the art. In the field, this is known as optoelectrowetting, and in the following we will refer to it by the corresponding acronym OE This is known as WOD and is disclosed, for example, in Patent Documents 1 to 4 and Non-Patent Document 1. In particular, the first of the four patent applications relates to a microstructure defined by first and second walls. The first wall is of composite design, including a fluid cavity, a substrate, a photoconductive layer and an insulating (dielectric) In this embodiment, a photoconductive layer and a photoelectron-transfer layer are provided. Disposed between the insulating layers is an array of conductive cells that are electrically insulated from one another and coupled to the photoactive layer. Its function is to position the corresponding electrowetting electrodes on the insulating layer. At these locations, the electrowetting field generates These photoconductive layers are capable of changing the surface tension characteristics of the droplets when light strikes them. This method makes switching easier and faster. Although this has the advantage of being able to perform this operation, it is somewhat limited by the arrangement of the electrodes. There are also limitations to the speed at which the droplet can be moved and the extent to which the actual droplet path can be changed.

[0004] A two-sided embodiment of this latter method is disclosed in the non-patent literature 2. In one example, The photopattern was deposited on a dielectric layer using a photoinduced bias on amorphous silicon. Using photoelectrowetting across the surface of Teflon AF, nanoparticles in the size range of 100–500 nm were fabricated. A device capable of manipulating relatively large droplets of up to m has been described. In the device described, the dielectric layer is thin (100 nm) and is located only on the walls of the photoactive layer. stomach.

[0005] Recently, in our pending application EP17177204.9, we have reported on the use of photoelectron emission to produce microdroplets. A device for manipulating a liquid droplet is described. In this OEWOD device, the liquid droplet is, for example, A microfluidic space defined by containing walls, such as a pair of parallel plates, that sandwich a small fluidic space. At least one of the encapsulation walls has a selective region for the semiconductor layer buried therein. A "virtual" electrowetting electrode is installed, which is generated by selectively irradiating light on the By selectively illuminating the layer with light from a separate light source, virtual electrowetting can be achieved. A virtual path of electrode positions can be temporarily generated and a microdroplet can be moved along it. The corresponding application EP17180391.9 describes the use of this device as an operating part of a nucleic acid sequencer. It is stated that:

[0006] Applicants currently provide, in some instances, a method for transferring microdroplets between different flow regions. This makes it possible, for example, to separate specific microdroplets into different regions. For example, chemical and enzymatic reactions occurring there can be incubated and captured. In another example, the carrier, fluid, or second emulsion may be added to the carrier or fluid for the purpose of An example of this latter is It is useful for cell culture, where microdroplets containing cells are held in place while dissolving A continuous phase flow containing nutrients and gas is passed over the droplets. This includes manipulating and testing male and female gametes during the IVF workflow. . [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 20030224528 [Patent Document 2] U.S. Patent Application Publication No. 20150298125 [Patent Document 3] U.S. Patent Application Publication No. 20160158748 [Patent Document 4] U.S. Patent Application Publication No. 20160160259 [Patent Document 5] PCT / EP2019 / 062791 [Non-patent literature]

[0008] [Non-Patent Document 1] Applied Physics Letters 93 221110, 2008 [Non-Patent Document 2] Pei, University of California, Berkeley paper UCB / EECS-2015-119 [Non-Patent Document 3] Spiegelaere et al. (2012). Methods Mol. Biol., vol 853, pp 29-37; Goossens et al. (2012). Anal. Biochem., vol 423(1), pp 93-101 Summary of the Invention [Problem to be solved by the invention]

[0009] According to the present invention, there is provided an apparatus for manipulating microdroplets, the apparatus comprising: A microfluidic chip adapted to receive and manipulate the dispersed microdroplets, The apparatus is characterized in that the flow path includes regions of different or zero flow of carrier fluid. . [Means for solving the problem]

[0010] In one embodiment of the invention, the microfluidic chip is adapted for, for example, electrowetting forces. The microdroplet includes one or more locations where the microdroplet is held in a stationary position by a retaining force, such as application of a force. In this embodiment, the electrowetting force employed is optically mediated (OE In yet another embodiment, the chuck is connected to a virtual electrode (WOD), which uses a virtual electrode of the type described above or below. The cup further comprises means for transferring the microdroplets between the various regions. The transport means may be a real or virtual object along which the or selected droplets may travel. Includes a path of electrowetting locations.

[0011] The droplets are attracted to the surface by being in an area of ​​low fluid flow or by (photo)electrochemical By being held by external forces such as wetting forces, or by the combination of the two aforementioned effects When the droplet is at rest due to the combination, the droplet is moved from its holding position without being displaced. In this way, it is possible to control the flow of the continuous phase using an external pump force. This has the advantage that the continuous phase can be exchanged around the droplet. The continuous phase contains dissolved gases and nutrients that are depleted by the metabolic activity of the encapsulated living cells. In the cell culture system, new substances are introduced from outside the microfluidic system to It is advantageous to replenish the depleted continuous phase. The pH of the droplet can be altered by displacing dissolved substances. Reagents such as carbon dioxide, which typically change the pH of the culture medium, are used to regulate the pH of the culture medium. In the case of the oxygen concentration, the carrier phase is externally controlled to be in equilibrium with the desired gas phase. The introduction can provide a transport pathway between the culture medium in the droplet and the gas phase.

[0012] The droplets held in the low flow area of ​​the chip are resuspended by flowing the carrier phase. The mechanism provided is that the carrier phase has a very high saturation capacity for solutes such as carbon dioxide and oxygen. It is particularly advantageous for the aqueous solution to have a relatively low saturation capacity for the aqueous material. This reduces the rate of dissolution of the water droplets into the oil phase, but increases the rate of dissolution of the water droplets from the continuous phase into the microdroplets. In this way, the replenishment of existing gases such as oxygen and carbon dioxide is carried out efficiently. Reduce the volume of the microdroplets containing cells without restricting access to the necessary gases This allows the cell population to be kept alive and proliferating within the microdroplets without the need for cell dispersion. .

[0013] When an analyte within the microdroplet dissolves in the continuous phase, the microdroplet is moved. The continuous phase can be allowed to flow without the need for a separatory filter to extract a sample of the analyte. It is also possible to introduce external reagents into the microdroplets using the flow of the continuous phase.

[0014] In an exemplary embodiment, the flow of the continuous phase is stopped by turning off the fluid pump and closing the valve. The cells cultured in the droplets secrete a compound, which is then terminated by The droplets naturally diffuse into the continuous phase. The spreading is promoted by agitating the droplets using a stirring force. A sample of the continuous phase that has accumulated the removed material is then reactivated by reactivating the pump and opening the associated valve. The process can also be reversed. Thus, material dissolved in the continuous phase can be fed to the droplets. This is a batch-flow format. Thus, a portion of the continuous phase can be introduced by the action of a fluid pump. The droplets are then incubated in the space around the droplets. The droplets and the liquid therein may be separated from the continuous phase by a constant flow. The uptake of substances into cells occurs by either passive diffusion, osmosis, or Ostwald ripening. This is carried out.

[0015] In addition to causing the flow of the continuous phase, the low flow area and the electrowetting force The second emulsion of microdroplets is then ejected from the outside of the chip while part of the previous droplet is stationary using the The droplets from the secondary emulsion are then transferred to the primary emulsion. The process is repeated with the tertiary emulsion. In this way, a series of different emulsions can be fed into the microfluidic chip using only a single inlet. Items can be brought into the pool one after another.

[0016] As mentioned above, in some embodiments, the present invention provides a method for the treatment of IVF in vitro fertilization workflow. The method can be applied to the manipulation and examination of male and female gametes.

[0017] For example, the device of the present invention can be used to examine male gamete cells, such as human or animal sperm cells. Screening, selection and assay procedures can be performed. In one example, sperm cells A sample of diluted semen is prepared and encapsulated in a droplet. The droplet is then attached to a chip, These droplets, which do not contain gametes, are discarded, while those containing sperm cells are examined under a bright-field microscope. The droplet is retained for examination. Once a sample of gametes to be analyzed is selected, the gametes are Video and still images of the child are captured. Pattern recognition algorithms are then applied to the video to By using these characteristics, it is possible to grasp the characteristics of gamete motility, body morphology, and nuclear morphology. The results of the sampling may be mapped to a particular droplet, which may then be collected and The treatment includes the addition of a reporter reagent, and other on-chip assays. It can also be used in in vitro fertilization processes and genetic analysis. Collection can also be done outside the facility.

[0018] In another example, the encapsulation of female gametes, such as human or animal eggs, results in fertilization of the eggs. Similar to male gametes, female gametes can be encapsulated in droplets and mounted on the chip. On the device, the cells can be examined for morphological defects or for the expression of reporter reagents. After examination or assay, the female gamete cells can be assayed using the movement of the droplets. The mechanical shear applied by the needle removes germinal epithelial cells and prevents the removal of additional reagents. The material may be optionally subjected to a processing step, such as addition of

[0019] In yet another example, male and female gametes can be integrated into a single microfluidic device. In one example, multiple gamete droplets can be merged together. A droplet of male gametes is combined with an egg, and the eggs are then mated on a chip via traditional gamete-gamete interactions. In another example, fertilization occurs between a single selected male gamete and a single selected Selected and processed single female gametes are then bound and allowed to interact on the chip.

[0020] In another application, gametes of both sexes are retrieved from the microfluidic chip and used for clinical trials such as ICSI or IVF. The bonding is accomplished using conventional processing techniques known in the art.

[0021] In some embodiments, the granules are shaped by the methods described above or by conventional methods known in the art. The generated blastocysts can also be encapsulated in droplets and cultured on the chip. The developing blastocyst can then be examined using the imaging and detection systems described below. In addition, by using droplet binding operations, buffer solutions, salts, nutrients, proteins, etc. can be This allows for control of the blastocyst's environment by adding materials such as extracellular matrix. During blastocyst formation, cells are extracted from the blastocyst using techniques such as laser microdissection. It is often desirable to remove samples of the cells and collect them for further analysis. In some embodiments, the blastocyst is delivered to a droplet manipulation area, which includes: Pillars, posts, physical limitations between electrowetting plates, or The gap between the electrowetting plates as described in Reference 5 The present disclosure may include physical features on the microfluidic chip, such as changes in the shape of a curve. Once the blastocyst is delivered to the manipulation area, the blastocyst is allowed to grow. The blastocysts are then held in a qualitatively immobile state. Laser microdissection can be performed as described in Once a portion of the droplet has been excised, the droplet splitting operations described herein can be used to separate the sample portion into a pre-extracted portion. The embryo can be separated from the blastocyst. The division and recombination process is repeated, and the two liquids after division are separated. The distribution of material between the droplets is checked by a machine visual inspection to ensure that the blastocyst and the sample portion are correctly aligned. After separation, the sample portion of the blastocyst is The samples were collected for further processing by genetic testing, including DNA sequencing and PCR. obtain.

[0022] As for the microfluidic chip itself, the various regions are optionally connected by a series of microfluidic pathways. The substrate may include, for example, one or more microfluidic channels, tubes, or other structures disposed on or between the walls of the substrate. In one embodiment, the optical detection system is preferably connected to the optical sensor. These paths include the locations of real or virtual electrowetting electrodes, The droplets are then moved along the electrodes by air pressure and / or electrowetting forces. In addition, various regions and optical detection systems can be implemented to accommodate many more such electrode positions. In another embodiment, the pathways may further include an in-plane constriction or an out-of-plane constriction. The in-plane constriction or the out-of-plane constriction may comprise a constriction in which the carrier phase is in contact with the in-plane constriction or the out-of-plane constriction. or out-of-plane constrictions, but the droplets can flow unhindered through the in-plane constrictions or Has dimensions that prevent it from passing through an out-of-plane constriction

[0023] In a preferred embodiment of the chip, the electrowetting electrodes include one or more O The microfluidic pathways and / or regions are virtually located by the EWOD structure. In this state, these OEWOD structures are - a first substrate; a first conductor layer on the first substrate having a thickness of 70 to 250 nm; Electromagnetic radiation in the wavelength range of 400-1000 nm, having a thickness in the range of 300-1500 nm, and a photoactive layer having a thickness of 30 to 160 nm formed on the photoactive layer. a first composite wall having a first dielectric layer having a second substrate, a second conductor layer having a thickness of 70 to 250 nm formed on the second substrate, and optionally A second dielectric layer having a thickness of 30 to 160 nm formed on the second conductor layer, The exposed surface of the second dielectric layer defines a microfluidic space adapted to contain said microdroplet. - a second composite wall arranged at least 10 m apart so as to be in contact with said first conductor layer and and an A / C source that supplies a voltage to the first composite wall and the second composite wall that connect the second conductor layer. , impinging on the photoactive layer to form a corresponding virtual electrowetting layer on the surface of the first dielectric layer; a band gap of the photoactive layer adapted to induce a position of a photodiode; at least one source of electromagnetic radiation having high energy; - manipulating the impingement point of electromagnetic radiation on the photoactive layer to generate the virtual electrowetting At least one of the poles along which the microdroplets can move by changing the arrangement of the pole positions. and means for forming an optically mediated electrowetting path.

[0024] In one embodiment, the first and second walls of the structure are transparent and the microfluidic cavity is disposed therebetween. In another embodiment, the first substrate and the first conductor layer are transparent; The light from the electromagnetic radiation source (e.g., multiple laser beams or LED diodes) In another embodiment, the second substrate, the second conductor layer, and The second dielectric layer is transparent, and thus can achieve the same purpose. In the embodiment, all of these layers are transparent.

[0025] Preferably, the first and second substrates are made of, for example, glass, metal, silicon, or The is made of a material having mechanical strength, such as engineering plastic. In this embodiment, the first substrate and the second substrate may have a certain degree of flexibility. In another embodiment, the first substrate and the second substrate have a thickness in the range of 100 to 1000 μm. .

[0026] The first conductor layer and the second conductor layer are formed on one surface of the first substrate and the second substrate. The thickness of the layer is typically in the range of 70 to 250 nm, and preferably in the range of 70 to 150 nm. At least one of the layers is made of a transparent conductive material such as indium tin oxide (ITO), a conductive material such as silver, etc. They are made of very thin films of conductive metals or conductive polymers such as PEDOT. These layers may be formed as a continuous sheet or may be a series of individual structures, such as wires. The conductive layer may be formed in a mesh shape, and the gaps in the mesh may be It is also possible to emit electromagnetic waves from

[0027] The photoactive layer is capable of generating a localized charge in response to electromagnetic stimulation. It is preferable that the semiconductor material is used. For example, an android In one embodiment, the photoactive layer comprises: It is activated using visible light.

[0028] The photoactive layer in the case of the first wall and, optionally, the conductive layer in the case of the second wall, are typically Typically, the substrate is covered with a dielectric layer having a thickness in the range of 30-160 nm. The dielectric properties of this layer are preferably 10 7High dielectric strength > 3 V / m and dielectric constant > 3, preferably to avoid dielectric breakdown In one embodiment, the dielectric layer is made of alumina, silica , hafnia, or a thin non-conductive polymer film.

[0029] In another embodiment of the structure, at least the first dielectric layer, or the second dielectric layer, preferably Either one or both of them are able to deliver the desired microdroplets / carrier liquid at the various virtual electrowetting electrode locations. / Helps establish a contact angle with the surface and further helps ensure that the contents of the microdroplets adhere to the surface. , are coated with an anti-fouling layer to prevent loss as they move through the chip. If the second wall does not include a second dielectric layer, the second antifouling layer may be applied directly onto the second conductor layer. For optimal performance, the antifouling layer should be measured at 250°C as an air-liquid-surface three-point interface. To establish the contact angle between the microdroplet / carrier liquid / surface, which should be in the range of 70-1100° when the In one embodiment, these layer(s) are less than 150 nm thick. In another embodiment, these layers have a triaxial structure, and may form a monolayer. Fluorocarbon-silanes such as perfluoro(1H,1H,2H,2H-perfluorooctyl)silane Preferably, either or both of the antifouling layers are included to ensure optimal performance. In certain embodiments, to form a chemically compatible interface between the layers, An interstitial layer of silica is interposed between the antifouling layer and the dielectric layer, the thickness of such layer being It is usually less than 10 nm.

[0030] The first and second dielectric layers, i.e., the first and second walls, are at least A microfluidic space having a width of 10 m is defined, in which the microdroplets are contained. The width of this space is 10 to 180 mm. The droplets have a characteristic diameter that is at least 10% and preferably at least 20% larger than the width of the microdroplet space. This causes the microdroplets that enter the chip to be compressed, and the electrons The wetting performance has been improved.

[0031] In one embodiment, the first and second dielectric layers are made of a protective material such as a fluorosilane. In another embodiment, the first dielectric layer and the front dielectric layer are coated with a stain-resistant coating. The second dielectric layer is made of (3-aminopropyl)trimethoxysilane, deposited proteins, collagen coated with a biocompatible coating such as a layer of fibronectin, laminin or fibronectin It has been done.

[0032] In another embodiment, the microfluidic space is configured to separate the first and second walls by a predetermined amount. The spacer includes one or more spacers for forming the spacer. The spacer includes one or more spacers formed by photopatterning. There can be beads, pillars, ridges, etc. made from an intermediate resist layer formed on the substrate. Use the pacer to create narrow channels, tapered channels, or pillar lines. By careful design, it is possible to form a partially enclosed channel defined by These spacers are used to aid in the deformation of the microdroplets and then Splitting can be performed and manipulations can be performed on the deformed microdroplets. The same spacer is used for priming and emptying the fluid in the microfluidic space. The flow can be induced.

[0033] The first wall and the second wall are biased using an A / C power supply attached to the conductor layer. A voltage potential difference in the range of 10 to 150 volts is applied between them.

[0034] These preferred OEWOD structures have wavelengths in the range of 400 to 1000 nm and a band gap of the photoexcitation layer. Preferably, the photoactivation is performed using a source of electromagnetic radiation having an energy higher than that of the photoactivation. The conductive layer is formed by applying the virtual energy when the incident intensity of the radiation used is in the range of 0.01 to 0.2 Wcm-2. In one embodiment, the electromagnetic wave source is activated at the location of the electrowetting electrode. A highly attenuated, and in another embodiment pixelated, photoexcitation region corresponding to said photoactive layer. This generates the pixelated virtual electrowetting electrode position is induced on the first dielectric layer.

[0035] When the source of the electromagnetic waves is pixelated, it is a digital microphotograph illuminated by the light of a lamp such as an LED. Directly or indirectly supplied by a reflective screen such as a small mirror device (DMD) This preferably forms a virtual electrowetting electrode on the first dielectric layer. This makes it possible to rapidly create and destroy complex patterns of electrode positions, By precisely controlling the impact force, the droplets can be precisely guided along essentially any virtual path. This means that the chip can guide thousands of microdroplets along multiple paths. This is particularly advantageous when simultaneous operation is required. The path of the electrowetting is determined by the location of a virtual electrowetting electrode on the first dielectric layer. The image output from a video microscope can be considered to be composed of a continuum. Using physical microfluidic channels patterned on the microdevice, By simultaneously inspecting both the projected and virtual electrowetting electrode position patterns, After this inspection, the position of the virtual electrowetting pattern is aligned with the fluid channel. The position of the lens can be precisely aligned to ensure proper alignment between the microfluidic and optical projector assemblies. Precisely transport droplets through various fluidic channels and flow regions without relying on mechanical adjustments. It can be sent.

[0036] The impingement point of the electromagnetic radiation source on the photoactive layer may be any convenient shape, including conventional circular and annular shapes. In one embodiment, the morphology of these points can be the shape of the corresponding pixelated morphology. and in another embodiment, the microdroplet after entering the microfluidic space is determined by In one preferred embodiment, the collision point, i.e. the front The electrowetting electrode position is crescent shaped and the intended location of the microdroplets is Preferably, the position of the electrowetting electrodes themselves faces the forward direction. The surface of the microdroplet attached to the first wall is smaller than the surface of the microdroplet, and the droplet and the dielectric of the surface are The maximum electric field gradient is provided across the contact line formed between the OEWOD structure. In an embodiment, the second wall also comprises a photoactive layer and is photoactivated by the same or a different electromagnetic radiation source. to induce the location of a virtual electrowetting electrode on the second dielectric layer. The addition of the second dielectric layer can be used to selectively wet the wetted edge of a given microdroplet. The structure can be transferred from the top to the bottom, and each microdroplet is then subjected to a large electrowetting A gripping force can be applied.

[0037] As mentioned above, the device is designed to investigate optical phenomena within or downstream of the chip. In one embodiment, the optical detection system may further include an optical detection system disposed as follows: The optical element is integrated with the chip and is located within a region where the flow of the microdroplets is zero. The detection system may, in one embodiment, be a bright field microscope, a dark field microscope, a chemiluminescence detection means, a Forster A preferred embodiment is selected from resonance energy transfer detection means and fluorescence detection means. In the present invention, the optical detection system is a means for stimulating and detecting the fluorescence of the microdroplets, and further, a detection region having an associated radiation-transmissive detection window, and an electromagnetic radiation irradiating said microdroplet. a light source (e.g., visible light, infrared light, or UV light), one or more light detectors, and optionally, and receiving a signal from the detector(s) to transmit the assay result or nucleotide sequence information, e.g. and a microprocessor that provides a visual display or count to the user. In one embodiment, the optical detection system detects a characteristic detection property associated with the microdroplet, preferably or directly or indirectly by interaction or reaction with the analyte being sought that is contained therein. Fluorescence from a reporter molecule (such as a biomarker or molecular beacon) that is activated It is designed to detect optical signals.

[0038] The device of the present invention comprises: (1) a carrier fluid that is immiscible, such as a fluorocarbon or silicone oil; (2) a means for generating a medium containing an emulsion of aqueous microdroplets in the body; A means for flowing through the chip from an inlet using a pump or mechanical injector, etc.; (3) Analytes or other biomolecules of the above types are stored in, for example, patient samples or cell culture incubators. a sample preparation region generated upstream of said inlet, such as from a sample preparation area

[0039] As mentioned above, in some cases, the saturation capacity for solutes such as carbon dioxide and oxygen is very low. By passing a carrier phase that is always large and has a relatively small saturation capacity for aqueous substances, It is advantageous to resupply the cells contained in the droplets.

[0040] Thus, the means (1) for generating the medium may be, for example, in a controlled atmosphere vessel. Incubating the vial of carrier phase and agitating it to ensure contact between the liquid phase and the gas phase. The carrier phase may further include a medium conditioning component for processing the carrier phase. Transfer the microfluidic nebulizer to a gas-impermeable sealed container (e.g., a glass syringe) and centrifuge the microfluidic nebulizer as described above. By pumping the liquid through a network, the dissolved gas is released by the respiration of the cells in the microdroplets. can replenish the depleted carrier phase.

[0041] In another example, the carrier phase stream is passed through a gas permeable tube or membrane and then passed through an equilibration vessel. Resupply is accomplished by exposure to a controlled atmosphere having the desired gas concentration. The gas diffuses from the atmosphere through the membrane into the carrier phase, increasing the gas concentration in the carrier phase. The flow path beyond the equilibration vessel is made up of permeable tubes, which are made of glass, molten steel, Place the device in a gas-impermeable tube, such as polyethylene, polyetheretherketone, or composite tube. Such a network allows the batch preparation of the support phase in a separate vessel. The treated carrier phase can be continuously supplied without the need for a separate equilibration vessel. The temperature is set between the gas bleed-in valve and the gas sensor arranged in the equilibrium container. The gas bleed valve may be controlled by a closed loop feedback system. allows gas to enter the vessel when the concentration measured by the sensor falls below a critical value. Alternatively, a continuous gas flow can be introduced through a flow regulating controller to achieve an equilibrium volume. The gas may be passed through the vessel at a flow rate selected to exceed the rate of gas depletion. It is explained as follows: [Brief description of the drawings]

[0042] [Figure 1] 1 shows an apparatus according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] As shown in FIG. 1, the apparatus according to the present invention first saturates fluorocarbon oil with carbon dioxide. The microfluidic tube 1 is inserted into a gaseous container 2 whose gas content is maintained at 5% carbon dioxide. The gas composition can be selected from the gas inlet and outlet. The fluorocarbon oil is then gasified and monitored by a carbon dioxide probe 5. The oil is then passed through the cavity via the permeable tube 6. The oil is then carbonated. The oil is delivered via a microfluidic tube 7 to a selector valve 8. The selector valve 8 contains a number of aqueous microdroplets. An emulsion 9 is provided, at least a portion of which is intended to be operated or detected by the user of the device. The microfluidic tube 10 is further connected to the 8, and the 8 setting Depending on the application, the composition may contain an emulsion, a fluorocarbon oil, or a mixture thereof.

[0044] Reference numeral 10 denotes a microfluidic device including a flow channel 12 in which an OEWOD virtual electrode (not shown) is provided, and a holding area 13. A droplet manipulator 11 is connected to the droplet manipulator 11. In use, the droplets flow through a flow path 12 to an output 13. is drawn from the channel 12 by applying a directional electrowetting force at the inlet 14. 13. In 13, the microdroplets are fluorocarbon oil microdroplets. The electrode may be held in place (not shown) to undergo electrowetting while traversing the In this state, the cells in the microdroplets can be efficiently cultured at the retention point. The drops are directed from 13 back to 12 and flow to 15 where they are collected for further processing and analysis.

Claims

1. An apparatus for manipulating microdroplets, comprising: adapted to receive and manipulate microdroplets dispersed in a carrier fluid flowing along a pathway a microfluidic chip having a The chip includes regions of different or zero flow of carrier fluid. An apparatus comprising:

2. 2. The apparatus of claim 1, wherein at least one region is configured to receive the microdroplets from the carrier stream. A device that is a holding area that is held in a stationary position in the body flow.

3. 3. The apparatus of claim 2, wherein the holding position is provided with an optically mediated holding force.

13. An apparatus comprising a barrier, well, or location where a barrier may be placed.

4. 4. The device according to claim 2 or 3, further comprising: a step of: transferring microdroplets to the holding area and 36. The apparatus of claim 35, further comprising a means for transporting the sample from the area.

5. 5. The apparatus according to claim 2, wherein the carrier fluid flow is dissolved therein. A device that contains gases, nutrients, biomolecules, or other chemical reagents.

6. 6. The apparatus of claim 5, wherein the material that dissolves in the flow of carrier fluid is A device that provides biological cells encapsulated within droplets with a local environment that promotes cell spreading.

7. 7. The device according to claim 1, wherein the chip comprises: a first substrate, a first conductor layer on said first substrate having a thickness of 70 to 250 nm, Electromagnetic radiation in the wavelength range of 400 to 1000 nm having a thickness in the range of 300 to 1500 nm formed on and a photoactive layer having a thickness of 30 to 160 nm formed on the photoactive layer. a first composite wall having a first dielectric layer having a second substrate, a second conductor layer having a thickness of 70 to 250 nm formed on said second substrate, and optionally a second dielectric layer having a thickness of 30 to 160 nm formed on the second conductor layer, The exposed surface of the second dielectric layer defines a microfluidic space adapted to contain said microdroplet. a second compound wall disposed at least 10 m away so as to - on the first and second composite walls connecting the first and second conductor layers an A / C source providing a voltage; impinging on the photoactive layer to form a corresponding virtual electrowetting pattern on the surface of the first dielectric layer; a band gap of the photoactive layer adapted to induce a position of a photodiode; at least one source of electromagnetic radiation having high energy; - Manipulating the impingement point of electromagnetic radiation on the photoactive layer to control the virtual electrowetting At least one of the poles along which the microdroplets can move by changing the arrangement of the pole positions. and a means for forming an optically mediated electrowetting path, comprising at least Both are essentially composed of a single OEWOD structure. An apparatus comprising:

8. 8. The apparatus of claim 7, wherein the first composite wall and the second composite wall each include a first composite wall and a second composite wall. The device further comprises a first antifouling layer and a second antifouling layer on the dielectric layer and the second dielectric layer. Place.

9. The device according to claim 7, further comprising a first antifouling layer on the first dielectric layer and / or the second dielectric layer. the second antifouling layer on the conductive layer is hydrophobic.

10. 10. The device according to claim 7, wherein the microfluidic space is formed by the first dielectric layer. and a spacer attached to the second dielectric layer.

11. An apparatus according to any one of claims 7 to 10, wherein the electrowetting path are hypothetical electrowetters, each of which may be exposed to the OEWOD at some point during use. a continuum of positioning positions,

12. The device according to any one of claims 7 to 11, wherein the microfluidic space comprises at least one The device is 10-80 μm in one dimension.

13. An apparatus according to any one of claims 7 to 12, comprising: the electromagnetic radiation source includes an array of pixelated lights; The light is reflected from or transmitted through the array. Device,

14. An optical detection system for detecting signals from the microdroplets located within or downstream of the chip. The device according to any one of claims 7 to 13, further comprising:

15. The device according to any one of claims 7 to 14, further comprising: The flow of the medium or immiscible carrier flow containing the emulsion of aqueous microdroplets flowing through the microfluidic chip is treated as an organic solvent. The apparatus further comprising:

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