Patent Grant
11857969
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Air-matrix digital microfluidic (DMF) apparatuses and methods for manipulating and processing encapsulated droplets are described herein.
Microfluidics-based technologies have proven useful in a wide variety of applications. While microfluidic manipulations are typically carried out using microchannels, an alternative paradigm has recently emerged, called digital microfluidics (DMF). In DMF, discrete nanoliter- (nL) to microliter-(μL) sized droplets of fluid are manipulated on a planar hydrophobic surface by applying a series of electrical potentials to an array of electrode pads. DMF has rapidly become popular for chemical, biological, and medical applications, as it allows straightforward control over multiple reagents, facile handling of both solids and liquids, and compatibility with even troublesome reagents (e.g., organic solvents, corrosive chemicals, etc.) because the hydrophobic surface is typically chemically inert.
Although DMF devices can handle different types of liquids, manipulating whole blood can cause a variety of difficulties, such as interfering with colorimetric assays and causing fouling. Further, many micro- and nano-fluidic assays are not capable of handling the often necessarily larger volumes of blood needed as the input to the assay directly. Therefore, it would be desirable to provide a DMF device that can extract plasma from a whole blood sample.
Described herein air-matrix digital microfluidic (DMF) methods for manipulating and processing blood, as well as apparatuses adapted to process blood.
We have recently developed a module for large-volume (milliliter-scale) sample extraction and concentration into the microliter volume used on the DMF device, utilizing a pre-fabricated cartridge and peristaltic pump to efficiently mix a sample with magnetic capture beads. To date, we have demonstrated microRNA extractions from up to 100 μL of plasma into a 2 μL droplet, with performance (recovery, quality) comparable to that achieved with bench-scale bead-based microRNA extraction. However, a continuing challenge for DMF is extracting plasma from whole blood for a complete sample-in-answer-out solution. In response to this challenge, we developed the first device architecture combining a plasma separation membrane from whole blood samples and downstream processing with DMF (see, e.g.,
For example, described herein are air-matrix digital microfluidic (DMF) apparatuses configured to process whole blood and manipulate plasma extracted from the whole blood. These apparatuses may include: a first plate having a first hydrophobic layer; a second plate having a first side coated with a second hydrophobic layer, the second plate having a sample outlet; an air gap formed between the first and second hydrophobic layers; a plurality of actuation electrodes adjacent to the first hydrophobic layer; a sample inlet positioned over the sample outlet, the sample inlet configured to receive a sample of whole blood; a plasma separation membrane positioned between the sample inlet and the sample outlet, the plasma separation membrane configured to extract plasma into the sample outlet from the whole blood in the sample inlet; and a controller programmed to actuate a subset of the plurality of actuation electrodes that are activated when the plasma extracted from the whole blood contacts the first plate in order to draw the plasma through the plasma separation membrane.
The sample inlet may have a hydrophobic or super-hydrophobic surface. The second plate may have a second side with a super-hydrophobic surface, wherein the plasma separation membrane is positioned between the super-hydrophobic surface of the second plate and the super-hydrophobic surface of the sample inlet. For example, the sample inlet may comprise a cover plate with a hole. The sample inlet may be positioned above the sample outlet such that when the sample of whole blood is placed in the sample inlet, gravity draws the plasma through the plasma separation membrane.
Any appropriate plasma separation membrane may be used. For example, the plasma separation membrane may be porous and has larger pores positioned towards the sample inlet and smaller pores positioned towards the sample outlet. The plasma separation membrane may be an assembly of a plurality of membranes having different pore sizes.
The first plate may be part of a reusable device and the second plate is part of a disposable cartridge. The actuation electrodes may be disposed on a removable film.
The sample outlet may be larger than the sample inlet.
Also described herein are methods of extracting plasma from whole blood in an air-matrix digital microfluidic (DMF) apparatus, the method comprising: introducing a sample of whole blood into a sample inlet of the air-matrix DMF apparatus; extracting plasma from the sample of whole blood in the sample inlet through a plasma separation membrane and into a sample outlet of the air-matrix DMF apparatus; transporting the extracted plasma from the sample outlet to one or more actuation electrodes of a plurality of actuation electrodes of the air-matrix DMF apparatus; and actuating the one or more actuation electrodes of the air-matrix DMF apparatus to actively extract plasma from the sample of whole blood.
The method may also include prewetting the plasma separation membrane before introducing the sample of whole blood into the sample inlet.
As mentioned, the sample inlet may be positioned above the sample outlet such that when the sample of whole blood is introduced into the sample inlet, gravity draws the plasma through the plasma separation membrane. The plasma separation membrane may be sandwiched between a pair of super-hydrophobic surfaces.
The extracted plasma may be transported from the sample outlet to one or more actuation electrodes at least in part by gravity.
The method may also include detecting when the extracted plasma contacts the one or more actuation electrodes. The method may also include actuating the one or more actuation electrodes after the extracted plasma contacts the one or more actuation electrodes.
The method may also include actuating the one or more actuation electrodes before the extracted plasma contacts the one or more actuation electrodes.
The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Described herein are air-matrix digital microfluidics (DMF) methods and apparatuses that may be used with a fresh or stored (e.g., frozen) blood same, including blood samples taken directly from a patient. An air-matrix DMF apparatus as described herein may be particularly useful for use with immediately processing blood samples as part of the DMF process.
In particular, described herein are air-matrix DMF apparatuses including a plasma separation membrane as part of the apparatus, including as part of a cartridge that may be applied to a DMF driving apparatus. The plasma separation membrane may be formed as part of the top (e.g., top surface, or top plate) of the DMF apparatus. The apparatus may be configured to enhance the capillary forces drawing plasma through the plasma separation membrane and into the air gap of the DMF apparatus. Without the enhancements described herein, the rate of flow of plasma through a typically membrane (e.g., filter, separation membrane, etc.) would be rate limiting and slow, and would further limit the usefulness of the apparatus for directly processing blood without the need for separation or other pre-treatments.
For example, in any of the apparatuses described herein, a plasma separation membrane may be included on the top plate of the digital microfluidic (DMF) apparatus. The apparatus may be configured to pre-wet the separation membrane and/or a method of using the apparatus may include prewetting the separation membrane, to enhanced capillary forces and achieve faster flow through membrane. The apparatus may be configured so that, upon contact of plasma with DMF surface, the electrode(s) is/are actuated to pull the plasma to the DMF device using electro wetting forces. For example, the apparatus may be configured to detect plasma contacting the one or more electrodes within a plasma loading region of the air gap, for example, by electrical detection (e.g., change of an electrical property of the electrode(s)), optical detection (e.g., an optical sensor aimed at the air gap region at or near the plasma loading region), etc. Once fluid, e.g., plasma, is detected within this region, the DM apparatus may electrically modify the electrowetting forces and move the droplet. Pulling the droplet away by adjusting the electrowetting force may increase the flow of plasma through the membrane and into the air gap.
In any of the apparatuses and methods described herein, the plasma separation membrane may be sandwiched between super hydrophobic surfaces. The loading region on the outward-facing side of the apparatus may be a super-hydrophobic surface (e.g., including super hydrophobic coatings). The super hydrophobic environment surrounding the membrane may prevent a blood sample from overflowing the edges of the separation membrane, and may help achieve a maximum volume flow through membrane.
Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.
In general, an air-matrix DMF apparatus as disclosed herein may have any appropriate shape or size. The air-matrix DMF apparatuses described herein generally include at least one hydrophobic surface and a plurality of activation electrodes adjacent to the surface; either the hydrophobic surface may also be a dielectric material or an additional dielectric material/layer may be positioned between the actuation electrodes and the hydrophobic surface. For example, in some variations, the air-matrix DMF includes a series of layers on a printed circuit board (PCB) forming a first or bottom plate. The outer (top) surface of this plate is the hydrophobic layer. Above this layer is the air gap (air gap region) along which a reaction droplet may be manipulated. In some variations a second plate may be positioned opposite from the first plate, forming the air gap region between the two. The second plate may also include a hydrophobic coating and in some variations may also include a ground electrode or multiple ground electrodes opposite the actuation electrodes. The actuation electrodes may be configured for moving droplets from one region to another within the DMF device, and may be electrically coupled to a controller (e.g., control circuitry) for applying energy to drive movement of the droplets in the air gap. As mentioned, this plate may also include a dielectric layer for increasing the capacitance between the reaction droplet and the actuation electrodes. The reaction starting materials and reagents, as well as additional additive reagents may be in reservoirs that may be dispensed into the air gap, where the reaction mixture is typically held during the reaction. In some instances the starting materials, reagents, and components needed in subsequent steps may be stored in separate areas of the air gap layer such that their proximity from each other prevents them from prematurely mixing with each other. In other instances, the air gap layer may include features that are able to compartmentalize different reaction mixtures such that they may be close in proximity to each other but separated by a physical barrier. In general, the floor of the air gap is in the first plate, and is in electrical contact with a series of actuation electrodes.
In some embodiments, one of the plates can be integrated into a reader device, and the other plate can be integrated into a removable, disposable cartridge, that when attached to the reader, form a two plate digital microfluidics system similar to that described herein. The reader device can be a permanent, reusable device that contains all or a bulk of the electronics for controlling the DMF system, and may optionally also containing sensors (i.e. sensors for measuring color and/or light, temperature or pH) for analyzing the droplets in the device. In addition, the actuation electrodes can be disposed on a film, which can also be made of a dielectric material. The film can be removably attached to one of the plates, such as the plate on the reader or the plate on the cartridge, while the other plate can have the ground electrode(s). For example, U.S. Pat. Nos. 8,187,864; 8,470,153; 8,821,705; 8,993,348; and 9,377,439, which are hereby incorporated by reference in their entireties, describe cartridge based DMF systems.
As shown, the disposable sheet 10 can be affixed to the electrode array 16 of the DMF device 14 with a back surface of the sheet 10 adhered or suctioned to the electrode array 16 in which the reagent depot 12 deposited on the surface of the sheet 10 (across which the reagent droplets are translated) is aligned with pre-selected individual electrode 18 of the electrode array 16 as shown in steps (1) and (2) of
After the reaction has been completed, the disposable film 10 may then be peeled off as shown in step (4) and the resultant reaction products 26 analyzed if desired as shown in step (5). A fresh disposable film 10 may then be attached to the DMF device 14 for the next round of analysis. The product 26 can be also analyzed while the removable substrate is still attached to the device DMF device 14. This process can be recycled by using additional pre-loaded substrates. In addition, the droplets containing reaction product(s) may be split, mixed with additional droplets, incubated for cell culture if they contain cells.
In some embodiments as shown in
The air gap DMF apparatuses described herein may also include other elements for providing the needed reaction conditions. For instance, the air gap DMF apparatuses may include one or more thermal regulators (e.g., heating or cooling element such as thermoelectric modules) for heating and cooling all or a region (thermal zone) of the air gap. In other instances, heating or cooling may be provided by controlling endothermic or exothermic reactions to regulate temperature. The air gap DMF apparatuses may also include temperature detectors (e.g., resistive temperature detector) for monitoring the temperature during a reaction run. In addition, the DMF apparatuses may also include one or more magnets that can be used to manipulate magnetic beads in an on demand fashion. For example, the magnet(s) can be an electromagnet that is controlled by a controller to generate a magnetic field that can agitate or immobilize magnetic beads.
Thus, the air gap DMF apparatuses described herein may include one or more thermal zones. Thermal zones are regions on the air gap DMF apparatuses (e.g., the air gap) that may be heated or cooled, where the thermal zones may transfer the heating or cooling to a droplet within the thermal zone through one or more surfaces in contact with the air gap region in the zone (e.g., the first plate). Heating and cooling may be through a thermal regulator such as a thermoelectric module or other type of temperature-modulating component. The temperature of one or many thermal zones may be monitored through a temperature detector or sensor, where the temperature information may be communicated to a computer or other telecommunication device. The temperature is typically regulated between 4° C. and 100° C., as when these apparatuses are configured to perform one or more reactions such as, but not limited to: nucleic acid amplifications, like LAMP, PCR, molecular assays, cDNA synthesis, organic synthesis, etc.
An air gap DMF apparatus may also include one or more thermal voids. Thermal voids may be disposed adjacent to the different thermal zones. The thermal voids are typically regions in which heat conduction is limited, e.g., by removing part of the plate (e.g., first plate) (forming the “void”). These voids may be strategically placed to isolate one thermal zone from another which allows the correct temperatures to be maintained within each thermal zone.
In general, any of the air-matrix DMF apparatuses described herein may include a separate reaction chamber that is separate or separable from the air gap of the apparatus, but may be accessed through the air gap region. The reaction chamber typically includes a reaction chamber opening that is continuous with the lower surface of the air gap (e.g., the first plate), and a reaction chamber well that forms a cup-like region in which a droplet may be controllably placed (and in some variations, removed) by the apparatus to perform a reaction when covered. The cover may be a mechanical cover (e.g., a cover the seals or partially seals the reaction chamber opening, or a cover that encapsulates, encloses or otherwise surrounds the reaction droplet, such as an oil or wax material that mixes with (then separates from and surrounds) the reaction droplet when the two are combined in the reaction chamber.
In general, the reaction chamber opening may be any shape or size (e.g., round, square, rectangular, hexagonal, octagonal, etc.) and may pass through the first (e.g., lower) plate, and into the reaction chamber well. In some variations, the reaction chamber opening passes through one or more actuation electrodes; in particular, the reaction chamber opening may be completely or partially surrounded by an actuation electrode.
In the air-matrix apparatuses described herein, the first (lower) plate may also include one or more reaction chamber openings (access holes) 105, 105′. Access to the reaction chamber wells may allow reaction droplets to be initially introduced or for allowing reagent droplets to be added later. In particular, one or more reaction droplets may be manipulate in the air gap (moved, mixed, heated, etc.) and temporarily or permanently moved out of the air gap and into a reaction chamber well though a reaction chamber opening. As shown, some of the reaction chamber openings 105′ pass through an actuation electrode. As will be shown in greater detail herein, the reaction chamber may itself include additional actuation electrodes that may be used to move a reaction chamber droplet into/out of the reaction chamber well. In some variations one or more actuation electrodes may be continued (out of the plane of the air gap) into the reaction chamber well.
In general, one or more additional reagents may be subsequently introduced either manually or by automated means in the air gap. In some instances, the access holes may be actual access ports that may couple to outside reservoirs of reagents or reaction components through tubing for introducing additional reaction components or reagents at a later time. As mentioned, the access holes (including reaction chamber openings) may be located in close proximity to a DMF actuation electrode(s). Access holes may also be disposed on the side or the bottom of the DMF apparatus. In general, the apparatus may include a controller 110 for controlling operation of the actuation electrodes, including moving droplets into and/or out of reaction chambers. The controller may be in electrical communication with the electrodes and it may apply power in a controlled manner to coordinate movement of droplets within the air gap and into/out of the reaction chambers. The controller may also be electrically connected to the one or more temperature regulators (thermal regulators 120) to regulate temperature in the thermal zones 115. One or more sensors (e.g., video sensors, electrical sensors, temperature sensors, etc.) may also be included (not shown) and may provide input to the controller which may use the input from these one or more sensors to control motion and temperature.
As indicated above, surface fouling is an issue that has plagued microfluidics, including DMF devices. Surface fouling occurs when certain constituents of a reaction mixture irreversibly adsorbs onto a surface that the reaction mixture is in contact with. Surface fouling also appears more prevalent in samples containing proteins and other biological molecules. Increases in temperature may also contribute to surface fouling. The DMF apparatuses and methods described herein aim to minimize the effects of surface fouling. One such way is to perform the bulk of the reaction steps in a reaction chamber that is in fluid communication with the air gap layer. The reaction chamber may be an insert that fits into an aperture of the DMF device as shown in
In general, having a dedicated reaction chamber within the DMF device minimizes surface fouling especially when the reaction is heated. Thus, while surface fouling may still occur within the reaction chamber, it may be mainly constrained to within the reaction chamber. This allows the majority of the air gap region floor to remain minimally contaminated by surface fouling and clear for use in subsequent transfer of reagents or additional reaction materials if needed, thus allowing for multi-step or more complex reactions to be performed. When the reaction step or in some instances, the entire reaction is completed, the droplet containing the product may be moved out of the reaction chamber to be analyzed. In some examples, the product droplet may be analyzed directly within the reaction chamber.
In order to bring the droplet(s) containing the starting materials and the reagent droplets into the reaction chamber, additional actuation electrodes, which may also be covered/coated with a dielectric and a hydrophobic layer (or a combined hydrophobic/dielectric layer), may be used.
In some variations contacts may penetrate the surfaces of the reaction chamber. For example, there may be at least ten electrical insertion points in order to provide sufficient electrical contact between the actuation electrodes and the interior of the reaction chamber. In other examples there may need to be at least 20, 30, or even 40 electrical insertion points to provide sufficient contact for all the interior surfaces of the reaction chamber. The interior of the reaction chamber may be hydrophobic or hydrophilic (e.g., to assist in accepting the droplet). As mentioned, an electrode (actuation electrode) may apply a potential to move the droplets into and/or out of the well.
In general, the actuation electrodes may bring the droplet into the well in a controlled manner that minimizes dispersion of the droplet as it is moved into the well and thus maintaining as cohesive a sample droplet as possible.
In instances where the reaction compartment is an independent structure integrated with the DMF devices as those shown in
In another embodiment, the electrodes embedded in the reaction compartments can include electrodes for the electrical detection of the reaction outputs. Electrical detection methods include but are not limited to electrochemistry. In some instances, using the changes in electrical properties of the electrodes when the electrodes contact the reaction droplet, reagent droplet, or additional reaction component to obtain information about the reaction (e.g., changes in resistance correlated with position of a droplet).
The apparatuses described herein may also prevent evaporation. Evaporation may result in concentrating the reaction mixture, which may be detrimental as a loss of reagents in the reaction mixture may alter the concentration of the reaction mixture and result in mismatched concentration between the intermediate reaction droplet with subsequent addition of other reaction materials of a given concentration. In some variations, such as with enzymatic reactions, enzymes are highly sensitive to changes in reaction environment and loss of reagent may alter the effectiveness of certain enzymes. Evaporation is especially problematic when the reaction mixture has to be heated to above ambient temperature for an extended period of time. In many instances, microfluidics and DMF devices utilize an oil-matrix for performing biochemical type reactions in microfluidic and DMF devices to address unwanted evaporation. One major drawback of using an oil matrix in the DMF reaction is the added complexity of incorporating additional structures to contain the oil.
The methods and apparatuses described herein may prevent or limit evaporation by the use of wax (e.g., paraffin) in minimizing evaporation during a reaction. A wax substance may include substances that are composed of long alkyl chains. Waxes are typically solids at ambient temperatures and have a melting point of approximately 46° C. to approximately 68° C. depending upon the amount of substitution within the hydrocarbon chain. However, low melting point paraffins can have a melting point as low as about 37° C., and some high melting point waxes can have melting points about 70-80° C. In some instances higher melting point waxes may be purifying crude wax mixtures.
As mentioned, wax is one type of sealing material that may be used as a cover (e.g., within a reaction chamber that is separate from the plane of the air gap). In some variations, wax may be used within the air gap. In particular, the wax may be beneficially kept solid until it is desired to mix it with the reaction droplet so that it may coat and protect the reaction droplet. Typically the wax material (or other coating material) may be mixed with the reaction droplet and enclose (e.g., encapsulate, surround, etc.) the aqueous reaction droplet.
When a reaction droplet is maintained within a paraffin coating, not only is evaporation minimized, but the paraffin may also insulate the reaction droplet from other potentially reaction interfering factors. In some instances, a solid piece of paraffin or other wax substance may be placed within a thermal zone of the air gap layer of the DMF device. For example, during a reaction, actuation electrodes may move a reaction droplet to a wax (e.g., paraffin) body. Upon heating to a melting temperature, the wax body may melt and cover the reaction droplet. The reaction then may continue for an extended period of time (including at elevated temperatures) without need to replenish the reaction solvents, while preventing loss by evaporation. For example wax-encapsulated droplet may be held and/or moved to a thermal zone to control the temperature. The temperature may be decreased or increased (allowing control of the phase of the wax as well, as the wax is typically inert in the reactions being performed in the reaction droplet). The temperature at that particular thermal zone may be further increased to melt the paraffin and release the reaction droplet. The reaction droplet may be analyzed for the desired product when encapsulated by the liquid or solid wax, or it may be moved to another region of the DMF device for further reaction steps after removing it from the wax covering. Paraffins or other wax materials having the desired qualities (e.g. melting point above the reaction temperature) may be used. For example, paraffins typically have melting points between 50 and 70 degrees Celsius, but their melting points may be increased with increasing longer and heavier alkanes.
Through this approach of enclosing a droplet in a shell of liquid wax, the reaction volume and temperature are maintained constant without the use of oil, a humidified chamber, off-chip heating, or droplet replenishment methods. Waxes other than paraffin can be used to prevent droplet evaporation as long as their melting temperature is higher than the ambient temperature, but lower or equal to the reaction temperature. Examples of such waxes include paraffin, bees and palm waxes. The wax-like solids can be thermally printed on the DMF device surface by screen-, 2D- or 3D-printing. This wax-mediated evaporation prevention solution is an important advancement in developing air-matrix DMF devices for a wide variety of new high-impact applications.
As mentioned, the wax-based evaporation methods described may be used in conjunction with the DMF devices having a reaction chamber feature, or they may be used without separate reaction chambers. When used within a reaction chamber, the wax may be present in the reaction chamber and the reaction droplet may be moved to the reaction chamber containing wax for performing the reaction steps requiring heating. Once the heating step has completed, the reaction droplet may be removed from the reaction chamber for detection or to perform subsequent reaction steps within the air gap layer of the DMF device.
In other embodiments, the wax may be liquid at room temperature or an oil can be used instead of a wax or a solid wax can be heated until it is liquid. Instead of a heated reaction zone with wax, the liquid wax or oil can be mixed with a reagent before introducing the mixture into the DMF device in order to prevent the reagent from evaporating. The reagent droplet will then have a liquid wax or oil shell surrounding the reagent, which can be manipulated as described above. In some embodiments, the liquid wax/oil can be added manually to the reagent by the user. In other embodiments, the liquid wax/oil and the reagent can be dispensed from reservoirs, mixed together, and introduced into the DMF device using a pump by the DMF device.
The methods and apparatuses described herein may be used for preventing evaporation in air-matrix DMF devices and may enable facile and reliable execution of any chemistry protocols on DMF with the requirement for a temperature higher than the ambient temperature. Such protocols include, but are not limited to, DNA/RNA digestion/fragmentation, cDNA synthesis, PCR, RT-PCR, isothermal reactions (LAMP, rolling circle amplification-RCA, Strand Displacement Amplification-SDA, Helicase Dependent Amplification-HDA, Nicking Enzyme Amplification reaction-NEAR, Nucleic acid sequence-based amplification-NASBA, Single primer isothermal amplification-SPIA, cross-priming amplification-CPA, Polymerase Spiral Reaction-PSR, Rolling circle replication-RCR), as well as ligation-based detection and amplification techniques (ligase chain reaction-LCR, ligation combined with reverse transcription polymerase chain reaction-RT PCR, ligation-mediated polymerase chain reaction-LMPCR, polymerase chain reaction/ligation detection reaction-PCR/LDR, ligation-dependent polymerase chain reaction-LD-PCR, oligonucleotide ligation assay-OLA, ligation-during-amplification-LDA, ligation of padlock probes, open circle probes, and other circularizable probes, and iterative gap ligation-IGL, ligase chain reaction-LCR, over a range of temperatures (37-100° C.) and incubation times (≥2 hr). Additional protocols that can be executed using the systems and methods described herein include hybridization procedures such as for hybrid capture and target enrichment applications in library preparation for new generation sequencing. For these types of applications, hybridization can last up to about 3 days (72 h). Other protocols include end-repair, which can be done, for example, with some or a combination of the following enzymes: DNA Polymerase I, Large (Klenow) Fragment (active at 25° C. for 15 minutes), T4 DNA Polymerase (active at 15° C. for 12 minutes), and T4 Polynucleotide Kinase (active at 37° C. for 30 minutes). Another protocol includes A-Tailing, which can be done with some or a combination of the following enzymes: Taq Polymerase (active at 72° C. for 20 minutes), and Klenow Fragment (3′→5′ exo-) (active at 37° C. for 30 minutes). Yet another protocol is ligation by DNA or RNA ligases.
Although the encapsulation of droplets in wax may prevent or reduce evaporation while executing chemistry protocols at elevated temperatures, after protocol completion, it has been discovered that when the droplet is removed and separated from the wax, e.g., by driving the droplet using the electrodes of the DMF apparatus, a small amount of liquid wax remains with the droplet as a coating even when the aqueous droplet is moved away from the wax, and that this wax coating may prevent or interfere with subsequent processing and analysis of the reaction droplet, particularly as the droplet cools and the wax solidifies around the droplet after the droplet is moved out of the heating zone. Therefore, in some embodiments, the wax encapsulated reaction droplet can be accessed through the wax coating using the systems and methods described herein, which enables facile and reliable execution of downstream biochemical processes.
To access the reaction droplet through the wax coating after the reaction droplet has been separated from the bulk liquid wax in the heating zone, an additional hydrophobic (e.g., oil) material may be added to the reaction droplet to help dissolve the solidified wax encapsulated the reaction droplet. For example, a carrier droplet (i.e., an aqueous droplet enclosed in a thin layer of oil) can be merged with the encapsulated reaction droplet. The carrier droplet gains access to the reaction droplet by having the oil from the carrier droplet dissolve and/or merge with the thin wax layer encapsulating the reaction droplet. Other materials other than oil may be used by the carrier droplet to break through the wax layer encapsulating the reaction droplet. For example, materials that are immiscible with aqueous reaction droplet and are capable of dissolving wax may be used, such as carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, diethyl ether, dimethyl formamide, ethyl acetate, heptane, hexane, methyl-tert-butyl ether, pentane, toluene, 2,2,4-trimethylpentane, and other organic solvents. Other materials that may be used to break through the wax layer include ionic detergents such as cetyltrimethylammonium bromide, Sodium deoxycholate, n-lauroylsarcosine sodium salt, sodium n-dodecyl Sulfate, sodium taurochenodeoxycholic; and non-ionic detergents such as dimethyldecylphosphine oxide (APO-10), dimethyldodecylphosphine oxide (APO-12), n-Dodecyl-β-D-maltoside (ULTROL®), n-dodecanoylsucrose, ELUGENT™ Detergent, GENAPOL® C-100, HECAMEG®, n-Heptyl β-D-glucopyranoside, n-Hexyl-b-D-glucopyranoside, n-Nonyl-b-D-glucopyranoside, NP-40 Alternative, n-Octanoylsucrose, n-Octyl-b-D-glucopyranoside, n-Octyl-b-D-thioglucopyranoside, PLURONIC® F-127, Saponin, TRITON® X-100, TRITON® X-114, TWEEN® 20, TWEEN® 80, Tetronic 90R4. At temperatures where a wax remains liquid, a carrier droplet encapsulated with wax may also be used to break through the wax encapsulating the reaction droplet. However, for lower temperatures where the wax solidifies, a carrier droplet coated with wax generally cannot be used since solid wax will prevent droplet movement.
For example,
As shown in
Because the reaction droplet may be surrounded by a thin layer of liquid wax 910 after separation from the heating zone 900, it may be difficult to merge the reaction droplet 908 with another aqueous droplet since the liquid wax 910 coating may act as a barrier. In addition, the liquid wax 910 may solidify as the droplet cools to form a physical barrier that impedes merger with another droplet. Therefore, to facilitate merging of a liquid wax 910 coated reaction droplet 908 or a cooled reaction droplet 908 with a solid wax coating with another droplet, a carrier droplet 912 can be used to merge with the reaction droplet 908 as shown in
After the carrier droplet 912 has been merged with the reaction droplet 908, further processing of the combined droplet 914 can proceed, such as extracting an analyte from the combined droplet 914 and/or perform other steps such as hybridizing capture probes, digesting the reaction product using an enzyme, amplifying the reaction product with a set of primers, and the like. For example, the carrier droplet 912 can be carrying beads for extracting the analyte, e.g., DNA or RNA or proteins. When the droplets are merged, the beads, which can be magnetic, can be used to mix the combined droplet 914 by application of a magnetic field. The target analyte binds to the beads, which can be immobilized against the substrate by the magnetic field to form a bead pellet 916, as shown in
To form the sample inlet 1002, a cover plate 1014, with a hole or port that can serve as the sample inlet 1002, can be placed over a hole or port in the top plate 1006 that can serve as the sample outlet 1004. The cover plate 1014 can be made of a hydrophobic or super-hydrophobic material or can be coated with a hydrophobic or super-hydrophobic layer 1016, as shown in
For example, in one embodiment, a cover plate 1014 having about a 1 mm to 10 mm ID hole (e.g. a 4 mm ID hole) can be spray-coated on both sides with a super-hydrophobic layer (e.g., ˜500 nm layer of NeverWet®) followed by post-baking in an oven (100° C., 10 min). The top plate 1006 of the DMF device 1000 can have about a 1 to 20 mm ID hole (e.g. a 10 mm ID hole) that is aligned with the hole in the cover plate 1014. The hole in the top plate 1006 may be larger than the hole in the cover plate 1014. For example, the hole in the top plate 1006 may be about 3 to 10 mm larger than the hole in the cover plate 1014. The top surface of the top plate 1006 that faces the cover plate 1014 can also be coated with a super-hydrophobic layer (as above) and the other side of the top plate 1006 with the ground electrode can be spin-coated with a hydrophobic layer (e.g., a 50 nm layer of Teflon-AF1600) followed by post-baking as above. The bottom plate 1008 of the DMF device 1000 can be fabricated from a six-layer PCB substrate bearing copper electrodes (e.g., a 43 μm thick layer) plated with nickel (e.g., a 185 μm thick layer) and gold (e.g. a 3.6 μm thick layer) that can be formed by conventional photolithography and etching techniques, and covered with dielectric tape (e.g. a 25 μm thick layer) or coating. The PCB substrate can have an array of electrodes, such as one-hundred and twenty actuation electrodes (e.g. each 3.5 mm×3.5 mm) with inter-electrode gaps of about 10 to 100 μm (e.g. 40 μm). The cover plate 1014 and top plate 100 can be assembled using screws, bolts, snaps, adhesives and/or other fasteners, with the separation membrane (e.g. PALL plasma separation membrane, Ann Arbor, MI) sandwiched in between. The bottom plate 1008 and top plate 1006 can be assembled with one or more spacers disposed between the two plates that separates the two plates by about 100 to 1000 μm (e.g. about 300 μm). For example, the spacer can be formed from one or more layers of double-sided tape (e.g. three pieces of double-sided tape having a total thickness of ˜300 μm). The double-sided tape can provide dual functions of spacing and fastening the top plate to the bottom plate.
As described above, in some embodiments, one of the plates can be integrated into a reader device, and the other plate can be integrated into a removable cartridge, that when attached to the reader, form a two plate digital microfluidics system similar to that described herein. In addition, the actuation electrodes can be disposed on a film, which can also be made of a dielectric material. The film can be removably attached to one of the plates, such as the plate on the reader or the plate on the cartridge, while the other plate can have the ground electrode(s). For example, the film can be attached to the PCB substrate of the bottom plate.
The process for extracting plasma from whole blood samples into the DMF device and onto the electrodes is depicted in
Once the plasma contacts the DMF surface with the actuation electrodes 1010, the actuation electrodes contacting the plasma and around the contact point are activated, thereby pulling the plasma towards the DMF surface using electrowetting forces, and then a volume between 10-250 μL (e.g., ˜70 μL) of the extracted plasma is actuated by actuation electrodes of the DMF device 1000 for further processing. In some embodiments, a sensor can be used for feedback control by detecting when the plasma contacts the bottom plate, and the actuation electrodes can be activated when the sensors detect the plasma on the plate. For example, the actuation electrodes and/or separate sensor electrodes can be used to measure capacitance, which changes when liquid covers the electrode. In some embodiments, the actuation electrodes 1012 below the sample outlet 1004 can be activated before the extracted plasma contacts the actuation electrodes and can be kept on until a sufficient amount of plasma has been extracted or can be kept on for a set or predetermined amount of time, such as about 1, 2, 3, 4, or 5 minutes. As mentioned above, one of the key features of the assembled architecture is the super hydrophobic environment surrounding the separation membrane 1012 which prevents or reduces the likelihood that blood sample overflows from the edge of the separation membrane and into the gap between the cover plate and top plate, which allows the DMF device to achieve a maximum or increased volume of plasma flow through the separation membrane. The systems and methods described herein result in extraction yields up to 2× the volume of plasma extraction from a given sample volume in comparison to benchtop lateral flow methods. Moreover, the quality of plasma collected using this DMF device is surprisingly comparable to plasma prepared by centrifugation and lateral-flow methods with respect to the degree of RBC hemolysis. The system is designed for facile reconfiguration and reprogramming, for accommodation of a wide range of blood volumes and plasma output.
DMF apparatuses that include embedded centrifuge tubes and/or well-plate wells (e.g.,
Prototype devices fabricated as described above performed better or as well as air-gap DMF apparatuses without reaction chambers.
To qualitatively evaluate the effect of wax bodies to prevent evaporation in our assays, loop mediated amplification (LAMP) reactions were executed while covered in liquid paraffin wax in tubes on the benchtop using a real-time PCR Machine. As shown in
Human Panel A beads from the TaqMan® miRNA ABC Purification Kit (Thermo Fisher Scientific). Aliquots of miRNA (4 ul), or “reaction droplets”, were loaded onto the DMF platform and brought to an array of electrodes overlaying the heating zone such that the droplet came into contact with the paraffin wall. The heating zone was then heated (65° C., 2 min) to melt the paraffin around the droplet. Once the paraffin melted, the reaction droplets were driven away from the heating zone and merged with miRNA Binding Beads (4×106 beads;
Cover plates bearing 4 mm ID hole were spray-coated on both sides with a super-hydrophobic layer (˜500 nm, NeverWet®) followed by post-baking in an oven (100° C., 10 min). Device top plates with 10 mm ID holes were coated with a super-hydrophobic layer (as above) on one side and the side comprising of ground electrode was spin-coated with a hydrophobic layer (50 nm, Teflon-AF1600) followed by post-baking as above. The bottom plate of the DMF device was designed in CAD systems, and Gerber files were outsourced to a third-party company for fabrication. Briefly, a six-layer PCB substrate bearing copper electrodes (43 μm thick) plated with nickel (185 μm) and gold (3.6 μm) were formed by conventional photolithography and etching 15, and covered with dielectric tape (25 μm). The substrate featured an array of one-hundred and twenty actuation electrodes (each 3.5×3.5 mm) with inter-electrode gaps of 40 μm. The cover and top plates were assembled by means of screws with the plasma separation membrane (PALL, Ann Arbor, MI) sandwiched in between. The bottom and top plates were assembled with a spacer consisting of three pieces of double-sided tape (total thickness of ˜300 μm).
A sample of whole blood (300 μL) was spotted directly onto a prewetted (with tris buffer) separation membrane. The sample was incubated for 3 minutes and during that time plasma transferred from the bottom of the separation membrane to the receiving DMF device surface by capillary forces of the receiving DMF surface. Once the plasma contacted the DMF surface, the actuation electrodes were activated, thereby pulling the plasma towards the DMF surface using electrowetting forces. Once a sufficient volume of plasma was collected (˜70 μL), the actuation electrodes were actuated by the DMF device for further processing of the collected plasma droplet.
When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.
Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”.
Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.
As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/−0.1% of the stated value (or range of values), +/−1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/−5% of the stated value (or range of values), +/−10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.
The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.
This application is a continuation of U.S. patent application Ser. No. 16/614,396, filed Nov. 18, 2019, titled “DIGITAL MICROFLUIDICS SYSTEMS AND METHODS WITH INTEGRATED PLASMA COLLECTION DEVICE,” now U.S. Pat. No. 11,413,617, which is a national phase application under 35 USC 371 of International Patent Application No. PCT/US2018/043293, filed Jul. 23, 2018, titled “DIGITAL MICROFLUIDICS SYSTEMS AND METHODS WITH INTEGRATED PLASMA COLLECTION DEVICE,” which claims priority to U.S. Provisional Patent Application No. 62/536,419, filed Jul. 24, 2017, titled “DIGITAL MICROFLUIDICS SYSTEMS AND METHODS WITH INTEGRATED PLASMA COLLECTION DEVICE,” each of which is herein incorporated by reference in its entirety for all purposes. This patent application may claim priority to International Application No. PCT/US2016/036015, titled “AIR-MATRIX DIGITAL MICROFLUIDICS APPARATUSES AND METHODS FOR LIMITING EVAPORATION AND SURFACE FOULING,” filed on Jun. 6, 2016.
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| Number | Date | Country | |
|---|---|---|---|
| 20230049633 A1 | Feb 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62536419 | Jul 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16614396 | US | |
| Child | 17888461 | US |
