Microfluidic device and method for loading fluid in microfluidic device
阅读说明:本技术 微流控器件以及在微流控器件中加载流体的方法 (Microfluidic device and method for loading fluid in microfluidic device ) 是由 艾马·杰恩·沃尔顿 莱斯利·安·帕里-琼斯 于 2019-09-12 设计创作,主要内容包括:一种微流控器件,包括:上基板和下基板,所述上基板和所述下基板间隔开,从而在所述上基板与所述下基板之间限定流体室;以及孔,用于将流体引入到所述流体室中;多个可独立寻址的阵列元件,每个阵列元件限定所述流体室的相应区域;以及控制装置,用于寻址所述阵列元件。所述控制装置被配置成:确定工作流体已被引入到所述流体室的第一区域中;以及向用户提供输出以指示工作流体存在于所述第一区域中。(A microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; and an aperture for introducing a fluid into the fluid chamber; a plurality of independently addressable array elements, each array element defining a respective region of the fluid chamber; and control means for addressing the array elements. The control device is configured to: determining that a working fluid has been introduced into a first region of the fluid chamber; and providing an output to a user indicating that a working fluid is present in the first region.)
1. A method of loading a fluid into a microfluidic device, the microfluidic device comprising:
an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; and
an aperture for receiving fluid into the fluid chamber;
the method comprises the following steps:
loading a filler fluid into the microfluidic device;
positioning a dispensing end of a fluid applicator at or near the aperture;
dispensing working fluid from the fluid applicator into a loading region adjacent the bore and external to the fluid chamber; and
forcing working fluid from the loading region into the fluid chamber through the aperture.
2. The method of claim 1, wherein forcing working fluid from the loading region into the fluid chamber comprises: dispensing a second fluid from the fluid applicator, thereby forcing working fluid from the loading region into the fluid chamber through the aperture.
3. The method of claim 2, wherein the second fluid is air, or wherein the second fluid is a filler fluid.
4. The method of any one of claims 1 to 3, wherein the microfluidic device is an electrowetting on active matrix dielectric AM-EWOD microfluidic device comprising a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element, and each array element defining a respective region of the fluid chamber;
and wherein the method further comprises: actuating at least one of the array elements of the microfluidic device to retain the dispensed working fluid in the fluid chamber of the microfluidic device.
5. A method according to claim 1, 2 or 3, wherein the microfluidic device is an AM-EWOD microfluidic device electrowetting on an active matrix medium, comprising a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element, and each array element defining a respective region of the fluid chamber;
and wherein forcing working fluid from the loading region into the fluid chamber comprises: actuating at least one of the array elements of the microfluidic device to draw the dispensed working fluid into the fluid chambers of the microfluidic device.
6. The method of claim 5, comprising: actuating at least one array element in a second region of the microfluidic device, the second region being located between the well and a target region of the microfluidic device for a working fluid.
7. The method of claim 6, wherein the second region of the microfluidic device has a width at its closest point to the well that is less than the width of the well.
8. The method of claim 7, wherein the second region comprises: a first portion having a width less than a width of the aperture; and a second portion having a second, greater width, a boundary between the first portion and the second portion being located between the aperture and a flow edge of the working fluid.
9. The method of claim 8, comprising: applying a time-varying actuation pattern such that a boundary between the first portion and the second portion moves away from the aperture as a flow edge of the working fluid moves away from the aperture.
10. The method of any of claims 6 to 9, further comprising: actuating a second set of array elements after the working fluid is detected in a second region of the fluid chamber.
11. The method of any of claims 6 to 10, further comprising: actuating the array element such that the second region of the fluid chamber matches the region of the fluid chamber occupied by the working fluid.
12. The method of any of claims 4 to 11, further comprising: actuating a target set of array elements of the microfluidic device, the target set of array elements corresponding to a target region of the fluid chamber, to move a working fluid introduced through the well toward the target region of the fluid chamber.
13. The method of claim 12, comprising: actuating the target set of array elements upon determining that the region of the fluid chamber occupied by working fluid has reached a predetermined size and/or upon determining that the rate of change of the size of the region of the fluid chamber occupied by working fluid is below a predetermined threshold.
14. The method of any of claims 8 to 13, further comprising:
determining a region where a working fluid has been introduced into the fluid chamber; and
providing an output to indicate the presence of the working fluid in the region.
15. A method of extracting fluid from an AM-EWOD microfluidic device, the microfluidic device comprising:
an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween;
a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element and each array element defining a respective region of the fluid chamber; and
an aperture for receiving fluid into the fluid chamber;
the method comprises the following steps:
extracting a working fluid from a first region of the microfluidic device, the first region being spaced from the well, by:
actuating one or more array elements of an AM-EWOD device to move working fluid from the first region to an unload region adjacent the aperture and outside the fluid chamber; and
removing working fluid from the unloading area into the fluid chamber via the aperture.
16. The method of claim 15, comprising: disposing a fluid applicator in the unload region prior to actuating the one or more array elements of the AM-EWOD device;
wherein removing working fluid from the unloading area comprises: removing working fluid from the unloading area with the fluid applicator.
17. The method of claim 16, comprising: actuating one or more array elements of a first region of the AM-EWOD device to retain a working fluid in the first region prior to disposing the fluid applicator in the unload region.
18. The method of claim 15, 16 or 17, wherein actuating one or more array elements of the AM-EWOD device to move working fluid from the first region to an unload region comprises: actuating at least one array element in a second region of the microfluidic device, the second region being located between the first region and the well.
19. The method of claim 18, wherein the second region of the microfluidic device has a width at its closest point to the well that is less than the width of the well.
20. The method of claim 19, wherein the second region comprises: a first portion having a width less than a width of the aperture; and a second portion having a second, greater width, a leading flow edge of the working fluid being located between the aperture and a boundary between the first portion and the second portion.
21. The method of claim 20, further comprising: a time-varying actuation pattern is applied such that as the flow edge of the working fluid moves toward the aperture, the boundary between the first portion and the second portion moves toward the aperture.
22. The method of any of claims 5 to 21, comprising: controlling a pattern of actuated array elements based on the sensed fluid locations in the microfluidic device.
23. The method of any of claims 5 to 22, comprising: the pattern of actuated array elements is controlled to divide the working fluid into two portions.
24. An electrowetting on active matrix dielectric AM-EWOD microfluidic device comprising:
an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; and
an aperture for introducing a fluid into the fluid chamber;
a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element, and each array element corresponding to a respective region of the fluid chamber; and
a control device for addressing the array elements, the control device being configured to:
determining that a working fluid has been introduced into a first region of the fluid chamber by controlling the EWOD array element to operate in a sensing mode; and
providing an output to a user to indicate that a working fluid is present in the first region;
wherein the control device is configured to actuate a first set of array elements of the microfluidic device, the first set of array elements corresponding to a first region of the fluid chamber, to move a working fluid introduced via the well toward the first region of the fluid chamber.
25. The device of claim 24, the control means configured to:
actuating a second set of array elements of the microfluidic device, prior to actuating the first set of array elements, the second set of array elements defining a second region of the fluid chamber different from the first region, the second region extending to the well.
26. The device of claim 25, the control means configured to: actuating the second set of array elements upon detection of a working fluid in a second region of the fluid chamber.
27. The device of claim 26, the control means configured to: actuating the second set of array elements such that the second region of the fluid chamber matches the region of the fluid chamber occupied by the working fluid.
28. The device of claim 27, the control means being configured to actuate the second set of array elements in a time-dependent manner.
29. The device of claim 27 or 28, the control means being configured to: upon determining that the area of the fluid chamber occupied by the working fluid has reached a predetermined size, actuating the first set of array elements.
30. The device of claim 27 or 28, the control means being configured to: actuating the first set of array elements upon determining that a rate of change of a dimension of a region of the fluid chamber occupied by a working fluid is below a predetermined threshold.
Technical Field
The present invention relates to a microfluidic device and to a method for loading a fluid into such a device. More particularly, the present invention relates to an electrowetting on active matrix dielectric (AM-EWOD) microfluidic device. Electrowetting on media (EWOD) is a known technique for manipulating fluid droplets on an array. Active matrix EWOD (AM-EWOD) refers to the implementation of EWOD in an active matrix array containing transistors, for example by using Thin Film Transistors (TFTs).
Background
Microfluidics is a rapidly expanding field that involves the manipulation and precise control of fluids on a smaller scale, typically handling sub-microliter volumes. There is an increasing interest in applying microfluidics to chemical or biochemical assays and synthesis, as well as to medical diagnostics ("lab-on-a-chip"), both in research and in production. In the latter case, the compact nature of such devices allows for rapid testing when much smaller clinical sample volumes need to be used than in conventional laboratory-based testing.
Microfluidic devices can be identified by the fact that: microfluidic devices have one or more channels (or more generally gaps) of at least one dimension less than 1 millimeter (mm). Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions, and various buffers. Microfluidic devices can be used to obtain various measurements of interest, including molecular diffusion coefficients, fluid viscosity, pH, chemical bonding coefficients, and enzyme reaction kinetics. Other applications of microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, enzymatic assays, flow cytometry, sample injection to analyze proteins via mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning, and chemical gradient formation. Many of these applications have been used for clinical diagnostics.
A number of techniques are known for manipulating fluids on a sub-millimeter scale, characterized mainly by laminar flow and the advantages of surface forces over volumetric forces. Most of the technologies fall into the category of continuous flow systems, which typically employ cumbersome external piping systems and pumps. Systems employing discrete droplets have the advantage of greater functional flexibility instead.
Electrowetting on media (EWOD) is a well known technique for manipulating discrete fluid droplets by applying an electric field. It is therefore a candidate for microfluidics for lab-on-a-chip technology. An introduction to the basic principles of this technology can be found in the following documents: "Digital microfluidics: is a true lab-on-a-chip possible? "(R.B. Fair, Microfluid Nanofluid (2007) 3: 245-281).
Fig. 1 shows a portion of a conventional EWOD device in cross-section. The device includes: the
An
The contact angle θ is defined as shown in FIG. 1, and is defined by a solid liquid (γ)SL) Liquid non-polar ambient fluid (gamma)LG) And a solid non-polar ambient fluid (gamma)SG) The balance of the surface tension components between the interfaces is determined and young's law is satisfied in the absence of an applied voltage, and the equation is given by:
in operation, a voltage referred to as the EW drive voltage (e.g., V in FIG. 1)T、VOAnd VOO) Applied externally to different electrodes (
Fig. 2 is a diagram depicting additional details of an exemplary AM-
As described above with respect to the representative EWOD structure, the EWOD channel or gap defined by the two substrates is initially filled with a non-polar filler fluid (e.g., oil).
Example configurations and operations of EWOD devices are described below. US6911132(Pamula et al, published 6/28/2005) discloses a two-dimensional EWOD array for controlling the position and movement of droplets in two dimensions. US6565727(Shenderov, published 5/20/2003) also discloses methods for other droplet operations, including splitting and merging droplets, and mixing droplets of different materials together. US7163612(Sterling et al, published 16.1.2007) describes how TFT-based thin film electronics can be used to control the addressing of voltage pulses to an EWOD array by using a circuit arrangement very similar to that employed in AM display technology.
Comments "Digital microfluidics: is a true lab-on-a-chip possible? "(R.B.Fair, Microfluid Nanofluid (2007) 3: 245-281) indicates that: the method of introducing the fluid into the EWOD device is not discussed in detail in the literature. It should be noted that this technique employs a hydrophobic inner surface. Therefore, it is often energetically disadvantageous to fill such devices with an aqueous fluid from the outside by means of a separate capillary action. Furthermore, this may still be the case when a voltage is applied and the device is in an actuated state. Capillary filling of non-polar fluids (e.g., oils) may be energetically favorable due to the lower surface tension at the liquid-solid interface.
There are some examples of miniature microfluidic devices that describe fluid input mechanisms. U.S. Pat. No.5,096,669(Lauks et al, published 1992, 3/17) shows a device that includes an inlet port and an access channel for sample input that is coupled to a balloon that pumps fluid around the device when actuated. This patent does not describe how to input discrete fluid droplets into the system, nor does it describe a method of measuring or controlling the input volume of such droplets. This control of the input volume (referred to as "metering") is important in avoiding overloading the device with excess fluid and aids in the accuracy of the measurements performed where a known volume or volume ratio is required.
US20100282608(Srinivasan et al; published 11/2010) describes an EWOD device comprising two portions with apertures through which fluid can enter. The patent does not describe how a fluid may be forced into the device, nor does it describe a method of measuring or controlling the input volume of such a fluid. The related application US20100282609(Pollack et al; published 11/2010) does describe a piston mechanism for inputting fluids, but also does not describe a method of measuring or controlling the input volume of such fluids.
US20100282609 describes the use of a piston to force fluid into a reservoir contained in an oil-containing device. US20130161193 describes a method of driving a fluid onto an oil-filled device by using, for example, a bi-stable actuator.
GB2542372 and WO2017/047082 describe a microfluidic AM-EWOD device configured to retain a metered volume of a fill fluid that partially fills a chamber, preferably in a portion of the chamber, when the chamber of the device contains the metered volume of the fill fluid. Figure 3 is a schematic plan view of the microfluidic AM-EWOD device of GB 2542372/WO 2017/047082 after a metered volume of fill fluid has been introduced into the fluid chamber. The metered volume of fill fluid does not completely fill the fluid chamber, and the portion of the fluid chamber containing the fill fluid is shown in phantom in fig. 3. The filler fluid is preferably retained in the first region 5 of the fluid chamber by a
Disclosure of Invention
A first aspect of the invention provides a microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; an aperture for introducing a fluid into the fluid chamber; and a fluid input structure disposed above the upper substrate and having a fluid well for receiving fluid from a fluid applicator inserted into the fluid well, the fluid well in communication with a fluid outlet disposed in the fluid input structure base, the fluid outlet adjacent to the aperture; wherein the fluid well comprises a first portion, a second portion, and a third portion, the first portion, the second portion, and the third portion being different from one another, the first portion of the well forming a reservoir for the filler fluid; the second portion of the well is configured to sealingly abut against an outer surface of the fluid applicator when the fluid applicator is inserted into the fluid well; and a third portion of the well is in communication with the fluid outlet, and a diameter of the third portion at an interface between the third portion and the second portion is greater than a diameter of the second portion at an interface between the third portion and the second portion. The microfluidic device may be an electrowetting on dielectric (EWOD) microfluidic device further comprising a plurality of element electrodes, each element electrode defining a respective element of the EWOD device.
In this regard, when inserting the fluid applicator into the fluid well, the portion of the fluid applicator that dispenses the working fluid (which is typically the end of the applicator) contacts the surface of the packing fluid in the well and enters the packing fluid in the well before the outer surface of the fluid applicator seals against the second portion of the well. This prevents air from being trapped in the working fluid dispensed from the applicator and thus from being introduced into the fluid chamber of the microfluidic device. (the term "under" refers to a device that is oriented as shown, for example, in FIG. 5(a) or 5 (b))
The second portion of the fluid well may be adjacent to the first portion of the fluid well. Alternatively, the second portion of the fluid well may be spaced apart from the first portion of the fluid well-for example, if the first portion has a different cross-section than the second portion, the first portion may be separated from the second portion by a "transition" portion in which the cross-section gradually changes from the cross-section of the first portion to the cross-section of the second portion to avoid abrupt changes in the cross-section of the fluid well.
The hole may be defined between the upper substrate and the lower substrate.
The hole may be defined in the upper substrate.
The axial length of the third zone of the well may be such that: when the fluid applicator is inserted into the fluid input structure such that the outer surface of the fluid applicator sealingly abuts the second portion of the well, the ends of the fluid applicator are spaced apart from the upper and lower substrates.
The fluid input structure may extend around a periphery of the upper substrate.
The device may include a plurality of apertures for introducing fluid into the fluid chamber; wherein the fluid input structure comprises a plurality of fluid wells, each fluid well being associated with a respective bore.
A second aspect of the invention provides a method of loading a fluid into the microfluidic device of the first aspect, the method comprising: loading a filler fluid into the microfluidic device such that the filler fluid at least partially fills a first portion of the fluid well; inserting a fluid applicator into the fluid well such that an outer surface of the fluid applicator is sealingly in abutting engagement with a second portion of the fluid well; and dispensing the working fluid from the fluid applicator.
In the method of this aspect, the portion of the fluid applicator from which the working fluid is dispensed (which is typically the end of the applicator, e.g., the tip of the applicator) is below the surface of the packing fluid in the fluid well when the outer surface of the fluid applicator is in sealing abutting engagement with the second portion of the well (and when the working fluid is subsequently dispensed from the applicator). This prevents air from being trapped in the dispensed working fluid and thus from being introduced into the fluid chamber of the microfluidic device.
The method may further comprise: a predetermined volume of working fluid is dispensed from the fluid applicator.
The method may further comprise: after the working fluid is dispensed from the fluid applicator into the fluid well, a second fluid is dispensed from the fluid applicator.
The dispensed second fluid may remain connected to the fluid applicator.
The second fluid may be a different fluid than both the filler fluid and the working fluid. The second fluid may be air.
The method may further comprise: at least one element electrode of the microfluidic device is actuated to retain the dispensed working fluid in a fluid chamber of the microfluidic device.
The method may further comprise: after actuating the at least one element electrode, a second fluid is drawn from the fluid chamber. This can be done by: the fluid applicator is removed from the well such that any second fluid dispensed from the fluid applicator into the microfluidic device is extracted upon removal of the applicator. For example, if the applicator is a pipette, dispensing the working fluid by pushing the pipette plunger to a first position (e.g., "stop" described below) and dispensing the second fluid by pushing the pipette plunger past "stop" in the manner described below, retracting the pipette from the well with the plunger held in the "down" position (where the pipette plunger is pushed to a maximum extent or at least still pushed past "stop") will cause the second fluid to retract from the chamber. This technique may be applied, if desired, in conjunction with one of the techniques described below for moving the dispensed working fluid to and/or maintaining movement of the dispensed working fluid in a "safe" region in the fluid chamber to eliminate (or substantially reduce) the risk of the working fluid being inadvertently withdrawn with the second fluid.
Alternatively, the withdrawal of the second fluid from the fluid chamber may be completed before the fluid applicator is retracted. For example, if the applicator is a pipette, dispensing the working fluid by pushing the pipette plunger to a first position (e.g., "stop" as described below) and dispensing the second fluid by pushing the pipette plunger past "stop" in the manner described below, leaving the pipette in place and returning the plunger to the stop position (or allowing the plunger to return to the stop position) will cause the second liquid to retract from the chamber. After the plunger has returned/is returned to the "off position and the second fluid is retracted, then the pipette may be retracted. This technique may be applied, if desired, in conjunction with one of the techniques described below for moving the dispensed working fluid to and/or maintaining movement of the dispensed working fluid in a "safe" region in the fluid chamber to eliminate (or substantially reduce) the risk of the working fluid being inadvertently withdrawn with the second fluid.
The method may further comprise: after actuation of the at least one element electrode, a volume of filler fluid is withdrawn from the fluid chamber. In the example where the applicator is a pipette and the second fluid is dispensed by pushing the pipette plunger "stop", allowing the pipette plunger to return to its "fully pulled" position and then retracting the pipette from the well will cause both the second fluid and the volume of filler fluid to be retracted from the chamber.
The volume of filler fluid drawn from the fluid chamber may be equal to the volume of working fluid dispensed from the fluid applicator.
The fluid applicator may be a pipette, and dispensing the fluid from the fluid applicator may include: advancing the plunger of the pipette to a first position to dispense the working fluid and subsequently advancing the plunger beyond the first position to dispense a second fluid; and withdrawing the second fluid from the fluid chamber may comprise: the fluid applicator is retracted from the well if the plunger exceeds the first position.
The fluid applicator may be a pipette, and dispensing the fluid from the fluid applicator may include: advancing the plunger of the pipette to a first position to dispense the working fluid and subsequently advancing the plunger beyond the first position to dispense a second fluid; and withdrawing the second fluid from the fluid chamber may comprise: the plunger is returned or allowed to return to the first position and then the fluid applicator is retracted from the well.
The method may further comprise: the area of the region of the fluid chamber where the working fluid is present is monitored as the second fluid and/or filler fluid is drawn. If the size of the region in which the working fluid is present should be reduced, this indicates that the working fluid has been inadvertently extracted, and an output may be provided to indicate this. In case of manual fluid loading, the output is provided to the user and may for example be an audible and/or visual output, whereas in case of automatic or robotic fluid loading, the output is provided to a control unit controlling said automatic or robotic fluid loading and may for example be an electrical or optical signal.
A third aspect of the invention provides a method of loading a fluid into a microfluidic device, the microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; an aperture for receiving fluid into the fluid chamber; and a fluid input structure disposed above the upper substrate and having a fluid well for receiving fluid from a fluid applicator inserted therein, the fluid well in communication with a fluid outlet disposed in the fluid input structure base, the fluid outlet adjacent the aperture, the method comprising: loading a filler fluid into the microfluidic device such that the filler fluid at least partially fills the fluid well; inserting the fluid applicator into the fluid well such that an outer surface of an end of the fluid applicator is sealingly in abutting engagement with the fluid well at a location below a surface of the packing fluid; and dispensing the working fluid from the fluid applicator into the fluid well.
The method may further comprise: a predetermined volume of working fluid is dispensed from the fluid applicator.
A fourth aspect of the invention provides an electrowetting on active matrix dielectric (AM-EWOD) microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; an aperture for introducing a fluid into the fluid chamber; a plurality of independently addressable array elements, each array element defining a respective region of the fluid chamber; and control means for said addressing of the array elements, said control means being configured to: by passingControlling the EWOD array elementOperating in a sensing mode, determining that a working fluid has been introduced into a first region of the fluid chamber; and providing an output to indicate that working fluid is present in the first region.
Once the working fluid is in the first region, the fluid applicator for dispensing the fluid may be removed without any risk of accidentally withdrawing the dispensed working fluid from the microfluidic device. Thus, in the case of manual loading of the working fluid, the output may inform the user that it is safe to remove the applicator, or in the case of automatic or robotic loading of the fluid, an output signal may be provided to a system controlling the automatic or robotic loading of the fluid so that the system may remove the fluid applicator.
The device of the fourth aspect may further comprise: a fluid input structure disposed above the upper substrate and having a fluid well for receiving fluid from a fluid applicator inserted into the fluid well, the fluid well in communication with a fluid outlet disposed in the fluid input structure base, the fluid outlet adjacent the aperture; wherein the fluid well comprises a first portion, a second portion and a third portion, the first portion of the well forming a reservoir for a filler fluid; a second portion of the well configured for sealing abutting engagement with an outer surface of a fluid applicator inserted into the fluid well; and a third portion of the well is in communication with the fluid outlet, and a diameter of the third portion at an interface between the third portion and the second portion is greater than a diameter of the second portion at an interface between the third portion and the second portion.
In the device of the first or fourth aspect, the control means may be configured to: actuating a first set of array elements of the microfluidic device, the first set of array elements corresponding to a first region of the fluid chamber to move a working fluid introduced via the well toward the first region of the fluid chamber.
In the device of the first or fourth aspect, the control means may be configured to: actuating a second set of array elements of the microfluidic device prior to actuating the first set of array elements, the first set of array elements defining a second region of the fluid chamber different from the first region, the second region extending to the well.
In the device of the first or fourth aspect, the control means may be configured to: actuating the second set of array elements upon detection of working fluid in a second region of the fluid chamber.
In the device of the first or fourth aspect, the control means may be configured to: actuating the second set of array elements such that the second region of the fluid chamber matches the region of the fluid chamber occupied by the working fluid.
In the device of the first or fourth aspect, the control means may be configured to: actuating the second set of array elements in a time dependent manner.
In the device of the first or fourth aspect, the control means may be configured to: upon determining that the area of the fluid chamber occupied by the working fluid has reached a predetermined size, actuating the first set of array elements.
In the device of the first or fourth aspect, the control means may be configured to: actuating the first set of array elements upon determining that a rate of change of a dimension of a region of the fluid chamber occupied by the working fluid is below a predetermined threshold.
A variation of the fourth aspect provides a microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; an aperture for introducing a fluid into the fluid chamber; and a plurality of independently addressable array elements, each array element defining a respective region of the fluid chamber. The device is configured to: determining that a working fluid has been introduced into a first region of the fluid chamber; and providing an output to a user indicating that a working fluid is present in the first region. Any of the features described herein as being suitable for use in the device of the fourth aspect may be provided in a device according to this variant of the fourth aspect.
A fifth aspect of the invention provides a method of loading a fluid into a microfluidic device, the microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; and an aperture for receiving fluid into the fluid chamber; the method comprises the following steps: loading a filler fluid into the microfluidic device; positioning an end of a fluid applicator at or near the aperture; dispensing working fluid from the fluid applicator into a loading region adjacent the bore and external to the fluid chamber; and forcing working fluid from the loading region into the fluid chamber through the aperture.
The method of this aspect may be used with a device in which the fluid may not be fully loaded into the desired region of the microfluidic device when the working fluid is initially dispensed from the fluid applicator.
Forcing or urging working fluid from the loading region into the fluid chamber may comprise: dispensing a second fluid from a fluid applicator, thereby forcing working fluid from the loading region into the fluid chamber via the aperture. In this embodiment, the fluid applicator is further actuated to dispense bubbles (or other fluid different from the dispensed working fluid) in order to fully load the working fluid into the desired region of the microfluidic device.
The second fluid may be a different fluid than the working fluid. The second fluid may be, for example, air, or may be a filler fluid.
The microfluidic device may be an active matrix electrowetting on dielectric (AM-EWOD) microfluidic device comprising a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element, and each array element defining a respective region of the fluid chamber; and the method may further comprise: actuating at least one of the array elements of the microfluidic device to retain the dispensed working fluid in the fluid chamber of the microfluidic device.
The microfluidic device may be an active matrix electrowetting on dielectric (AM-EWOD) microfluidic device comprising a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element, and each array element defining a respective region of the fluid chamber; and wherein forcing working fluid from the loading region into the fluid chamber may alternatively or additionally comprise: actuating at least one array element of the microfluidic device to draw the dispensed working fluid into the fluid chambers of the microfluidic device.
The method can comprise the following steps: actuating at least one array element in a second region of the microfluidic device, the second region being located between the well and a target region of the microfluidic device for a working fluid. Whether the array element or elements are actuated depends on, for example, the reference of the drop being processed and/or the configuration of the EWOD device, particularly the relative values of cell gap, electrode size and drop size
The width of the second region of the microfluidic device at its closest point to the well may be less than the width of the well. (in many cases the second region will extend to and possibly through and into the port, in which case the second region of the microfluidic device will have a width at the port that is less than the width of the port
The second region may include: a first portion having a width less than a width of the aperture; and a second portion having a second, greater width, and a boundary between the first portion and the second portion may be located between the aperture and a flow edge of the working fluid. (Note that the first and second portions of the second region are defined by actuation of the array elements of the EWOD device, and that the boundary between the first and second portions is a conceptual boundary rather than a physical boundary.)
The method can comprise the following steps: applying a time-varying actuation pattern to an array element of the EWOD device such that a boundary between the first portion and the second portion moves away from the aperture as a flow edge of the working fluid moves away from the aperture.
The method may further comprise: a target set of array elements corresponding to a target region of the fluid chamber is actuated to move the working fluid introduced through the aperture to the target region of the fluid chamber. Likewise, a "target" region is a region of the fluid chamber into which it is desired to load a working fluid.
The method may further comprise: prior to actuating the target set of array elements, actuating a second set of array elements of the fluid chamber, the second set of array elements defining a second region of the fluid chamber different from the target region, the second region being closer to the aperture than the target region. In this embodiment, the second set of array elements is actuated to assist in initially loading the working fluid into the microfluidic device and/or to assist in initially moving the working fluid to a target area of the working fluid. Subsequently, the second set of array elements is de-actuated and the target set of array elements is actuated to assist in completing the movement of the working fluid to the target region of the working fluid.
The method may further comprise: the second set of array elements is actuated upon detection of the working fluid in the second region of the fluid chamber (e.g., in response to detection of the working fluid in the second region of the fluid chamber) or after detection of the working fluid in the second region of the fluid chamber.
The method may further comprise: the array element is actuated such that the second region of the fluid chamber matches the region of the fluid chamber occupied by the working fluid.
The method may further comprise: actuating a target set of array elements of the microfluidic device, the target set of array elements corresponding to a target region of the fluid chamber, to move a working fluid introduced through the well toward the target region of the fluid chamber.
The method may further comprise: the second set of array elements is actuated in a time dependent manner.
The method may further comprise: the target set of array elements is actuated upon (or after) determining that the area of the fluid chamber occupied by the working fluid has reached a predetermined size, and/or upon (or after) determining that the rate of change of the size of the area of the fluid chamber occupied by the working fluid is below a predetermined threshold.
The method may further comprise: determining a region where the working fluid has been introduced into the fluid chamber; and providing an output to indicate the presence of the working fluid in the region. For example, the region may be a target region of a fluid chamber into which it is desired to load working fluid, in which case the output indicates that working fluid has been successfully loaded into the target region of the fluid chamber. Alternatively, the region may be a region of the fluid chamber into which it is not desired to load working fluid, in which case the output indicates that an error has occurred in loading the working fluid. In case of manual fluid loading, the output is provided to the user and may for example be an audible and/or visual output, whereas in case of automatic or robotic fluid loading, the output is provided to a control unit controlling said automatic or robotic fluid loading and may for example be an electrical or optical signal.
Alternatively or additionally, the method may further comprise: the method includes determining a region of the fluid chamber into which the working fluid has been introduced, comparing the region to a desired region, and providing an output based on the comparison. For example, the method may provide an output (alarm) if the region into which the working fluid has been introduced is different from the region into which it is desired to introduce the working fluid. For example, if the area occupied by the working fluid is less than the area into which the working fluid is desired to be introduced, this indicates that an insufficient amount of working fluid is introduced, and if the area occupied by the working fluid is greater than the area into which the working fluid is desired to be introduced, this indicates that an excessive amount of working fluid is introduced. Alternatively, if the region occupied by the working fluid has the same area as the region into which the working fluid is desired to be introduced, but is displaced (partially overlapping or separated) relative to the region into which the working fluid is desired to be introduced, this is indicative of fluid being introduced into an incorrect region of the device.
Alternatively or additionally, the method may further comprise: the fluid chamber is monitored for the presence of the working fluid as the fluid applicator is withdrawn. If the size of the region in which the working fluid is present should be reduced, this indicates that the working fluid has been inadvertently withdrawn, and an output may be provided to alert the user/control unit. However, if the size of the region in which the working fluid is present does not decrease as the fluid applicator is withdrawn, this indicates that the fluid applicator was successfully withdrawn without causing the working fluid to retract from the fluid chamber, and an output confirming this may alternatively or additionally be provided.
In the method of the fifth aspect, the device may further include: a fluid input structure disposed above the upper substrate and having a fluid well for receiving fluid from a fluid applicator inserted into the fluid well, the fluid well in communication with a fluid outlet disposed in the fluid input structure base, the fluid outlet adjacent the aperture; wherein the fluid well comprises a first portion, a second portion and a third portion, the first portion of the well forming a reservoir for a filler fluid; a second portion of the well configured for sealing abutting engagement with an outer surface of a fluid applicator inserted into the fluid well; and a third portion of the well is in communication with the fluid outlet and has a diameter at an interface between the third portion and the second portion that is greater than a diameter of the second portion at an interface between the third portion and the second portion; and the method may comprise: loading a filler fluid into the microfluidic device prior to dispensing a working fluid from a fluid applicator such that the filler fluid at least partially fills a first portion of the fluid well; and inserting the fluid applicator into the fluid well such that the outer surface of the fluid applicator sealingly engages against the second portion of the fluid well.
A sixth aspect of the invention provides a method of extracting fluid from an AM-EWOD microfluidic device, the microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber therebetween; a plurality of independently addressable array element electrodes, each array element electrode defining a respective array element and each array element defining a respective region of the fluid chamber; and an aperture for receiving fluid into the fluid chamber; the method comprises the following steps: extracting a working fluid from a first region of the microfluidic device, the first region being spaced from the well, by: actuating one or more array elements of an AM-EWOD device to move working fluid from the first region to an unload region adjacent the aperture and outside the fluid chamber; and removing working fluid from the unloading area into the fluid chamber via the aperture.
The method of the sixth aspect may comprise: disposing a fluid applicator in the unload region prior to actuating the one or more array elements of the AM-EWOD device; wherein removing working fluid from the unloading area comprises: removing working fluid from the unloading area with the fluid applicator.
The method of the sixth aspect may comprise: actuating one or more array elements of a first region of the AM-EWOD device to retain a working fluid in the first region prior to disposing the fluid applicator in the unload region.
Actuating one or more array elements of the AM-EWOD device to move working fluid from the first region to an unload region may comprise: actuating at least one array element in a second region of the microfluidic device, the second region being located between the first region and the well. Whether the array element or elements are actuated depends on, for example, the volume of the droplet being processed and/or the configuration of the EWOD device, particularly the relative values of cell gap, electrode size and droplet size.
The width of the second region of the microfluidic device at its closest point to the well may be less than the width of the well. (in many cases the second region will extend to and possibly through and into the port, in which case the second region of the microfluidic device will have a width at the port that is less than the width of the port
The second region may include: a first portion having a width less than a width of the aperture; and a second portion having a second, greater width, a leading flow edge of the working fluid being located between the aperture and a boundary between the first portion and the second portion. (Note that the first and second portions of the second region are defined by actuation of the array elements of the EWOD device, and the boundary between the first and second portions is not a physical boundary, but is only defined by a varying activation pattern applied to the array elements.)
The method of the sixth aspect may further comprise: a time-varying actuation pattern is applied such that as the flow edge of the working fluid moves toward the aperture, the boundary between the first portion and the second portion moves toward the aperture.
The method of the fifth or sixth aspect may comprise: controlling a pattern of actuated array elements based on the sensed fluid locations in the microfluidic device. Alternatively, other methods may be used, for example, applying a predetermined time-varying actuation pattern.
The method of the fifth or sixth aspect may comprise: the pattern of actuated array elements is controlled to divide the working fluid into two portions.
In any aspect or embodiment, the microfluidic device may be an electro-wetting on Dielectric (EWODEl) device.
Drawings
Preferred embodiments of the invention will now be described by way of illustrative examples with reference to the accompanying drawings, in which:
fig. 1 is a cross-sectional view depicting a conventional EWOD device.
Fig. 2 is a schematic perspective view depicting an exemplary AM-EWOD device.
Fig. 3 is a schematic top view of the microfluidic device described in WO 2017/047082.
Fig. 4 is a schematic perspective view of a housing for a microfluidic device according to an embodiment of the present invention.
Fig. 5(a) is a partial cross-sectional view of a microfluidic device having a housing as shown in fig. 4.
Fig. 5(b) corresponds to fig. 5(a), but shows the pipette inserted.
Fig. 6 (a) to (f) are schematic top views of a microfluidic device illustrating a method of loading a fluid into the device according to one embodiment of the present invention.
Fig. 7 (a) to (f) are schematic top views of a microfluidic device illustrating a method of loading a fluid into the device according to another embodiment of the present invention.
Fig. 8 is a plan view of an AM-EWOD device illustrating a method of fluid loading.
Fig. 9 is a plan view of an AM-EWOD device illustrating another fluid loading method.
Fig. 10 (a), (b) and (c) are plan views of AM-EWOD devices, showing another fluid loading method.
Fig. 11 is a plan view of an AM-EWO device illustrating a method of fluid extraction.
Fig. 12 (a), (b), and (c) are plan views of AM-EWOD devices, illustrating another fluid loading method.
Fig. 13 illustrates a technique that may be applied to fluid loading or fluid extraction.
Detailed Description
Accordingly, embodiments of the present invention will now be described with reference to the drawings, wherein like reference numerals may be used to refer to like elements throughout. It should also be understood that the drawings are not necessarily to scale.
It has been recognised that whilst the microfluidic device of GB 2542372/WO 2017/047082 shown in figure 3 facilitates loading of the working fluid (also referred to as the "assay fluid" or "aqueous fluid") into the fluid chamber, two problems may arise in any subsequent heating of the device (as required in certain applications of such a device).
One problem that may arise in the device of fig. 3 is that if the total volume of fluid (filler fluid and working fluid) loaded into the fluid chamber is less than the total volume of the fluid chamber of the device, bubbles of air (or other exhaust fluid) will remain within the device. As long as the device is maintained at a uniform temperature (e.g., at room temperature), and the cell gap of the device is relatively uniform, the bubble will remain in a controlled position in
In principle, this problem can be avoided by: it is ensured that a precise volume of filler fluid is loaded into the device so that all of the exhaust fluid is expelled from the device when the working fluid is loaded, or is topped up with filler fluid after loading of the working fluid is complete. The first approach is however difficult to implement in practice, since small variations in device capacity and pipetting volume are unavoidable. The second approach is acceptable for laboratory use, but is not necessarily an ideal aspect for commercial products intended for non-laboratory conditions.
A second problem that may arise in the device of fig. 3 is that even if all of the required fluid is loaded into the device, with a single step of loading the oil (or other filler fluid) and no air bubbles remaining, the oil (or other filler fluid) will evaporate into the atmosphere upon heating of the device. This reduces the fluid volume in the fluid chamber and the air bubbles reappear.
One solution to the first problem is to fill the fluid chamber with the filler fluid at a first stage of the fluid loading process and then load the working fluid into the fluid chamber while the device is filled with the filler fluid. The method for achieving this is as follows. However, this does not solve the second problem because air bubbles may reappear after heating the device, and thus this approach is limited to situations where the device is not heated non-uniformly.
It has been found that completely sealing the device to prevent the filler fluid from evaporating is not a solution because if the device is heated, any air gaps between the seal and the filler fluid will expand and these expanding air bubbles may then encroach on the active area of the device.
1. Loading working fluid through housing
Fig. 4 shows a
The housing contains at least one fluid well 62 and preferably a plurality of fluid wells. Fig. 5(a) is a cross-section of a microfluidic device having a
Furthermore, it may be advantageous to use a fluid applicator that can dispense a predetermined amount of working fluid; and it is particularly advantageous to use a fluid applicator that can be loaded with a precise amount of fluid that it is desired to dispense so that no working fluid remains in the applicator after a predetermined amount of working fluid has been dispensed.
Fig. 5(b) corresponds to fig. 5(a), but shows the dispensing
The housing may be manufactured by any suitable process, for example by plastic injection moulding or by 3D printing. The microfluidic device may then be placed in and attached to a housing, and the resulting product is sometimes referred to as a "cartridge". The housing and the microfluidic device may be attached together in any suitable manner (e.g., using an adhesive). In one method of manufacture described in co-pending european patent application No. 18182737.9, the contents of which are incorporated herein by reference, the substrate of the microfluidic device is initially attached to the housing using double-sided adhesive tape.
Once the housing is checked to be correctly positioned, additional adhesive may be introduced into the joint between the housing and the substrate of the microfluidic device, e.g. by capillary filling, to ensure a fluid-tight seal between the housing and the substrate.
Fig. 4 shows a
In fig. 5(a), it can be seen that the fluid well/pipette port includes 4 main areas. These areas are arranged in succession along the axis of the fluid well, with the
The
The second region 2 serves as a transition between the first region 1 (wide) and the third region 3 (narrow).
The third region 3, or "sealing region", is a small diameter region (the cross-sectional diameter of the well in the third region is smaller than the cross-sectional diameter in the first region) which is used to form a seal with the end of the pipette when the pipette is introduced into the fluid well (and reasonably firmly pushed down). The taper angle of the wall in the third region 3 preferably matches the taper of the pipette tip to form a safety seal that exists over a range of heights, rather than only at one height (as would be the case if the angle were different from the angle of the tip of the pipette). (alternatively, if the pipette or other applicator is made of a material that deforms when inserted into the well, a secure seal may be obtained even if the taper angle of the third region does not match the taper angle of the pipette/applicator; in which case the third region may have a zero taper angle, and thus a substantially uniform cross-section over its length.)
As described below, in a preferred method of loading a working fluid into a fluid chamber, the level of filler fluid within the microfluidic device is sufficiently high while the working fluid is being loaded such that the filler fluid extends at least partially into the second region 2 and possibly into the
In principle, the third region 3 may extend all the way to the end of the port. However, if the housing is to be made by injection moulding, the minimum diameter of any hole is about 1 mm. Since the end of most commercial pipette tips have a smaller diameter than this, the
The
The third region 3 has a cross-section complementary to the outer cross-section of the dispensing
As noted, fig. 5(a) shows a housing suitable for use with a "side-loading" microfluidic device, wherein
It should be understood that fig. 5(a) shows one embodiment of a fluid well, but variations may be made. As an example, it is in principle possible to omit the second region 2 and to make the floor of the
In the embodiment of fig. 4, the housing extends around the entire periphery of the upper substrate when the microfluidic device is placed in the housing. In principle, however, the housing in general, and the fluid input port in particular, need not extend around the entire periphery.
In the embodiment of fig. 4, the housing contains a plurality of fluid wells. Microfluidic devices typically contain a plurality of wells for loading fluid into the device, and when the microfluidic device is placed in a housing, some or all of the fluid wells will be adjacent to corresponding fluid loading wells of the device. In general, there may be one or more wells for loading the packing fluid and one or more wells for loading the working fluid. Preferably, each well for loading the working fluid has a cross-section as shown in fig. 5 (a); the well for loading the packing fluid may have a cross-section as generally shown in fig. 5(a), or may have another cross-section.
In embodiments of the device as described with respect to fig. 4 and 5, the inner diameter of region 3 at the interface with
Certain examples of methods of use of these pipette ports will now be described.
Method 1-fast fluid infusion
In a first method of use, a pipette (or other fluid applicator) is loaded with working fluid as usual and then inserted into the associated fluid well. The housing and the microfluidic device have been loaded with the filler fluid such that the level of the filler fluid is located in the
It can thus be seen that the fluid well of this aspect has the following advantages:
the fluid well may be filled with oil (or other filler fluid) when initially loaded with filler fluid;
the wells form a reserve for the filler fluid and thus avoid the need for a dedicated reserve that takes up valuable space around the device;
the working liquid can be successfully loaded even when the microfluidic device is filled with the filler fluid.
In the case of a manual pipette, as described above, one way to dispense working fluid after insertion of the pipette is then for the user to slowly push the pipette plunger down from its "fully withdrawn" position, first to the normal "stop" so that working fluid flows from the end of the pipette into the
In other embodiments of the method, the electrodes of the device may be controlled to further ensure that working fluid loaded into the device is not inadvertently withdrawn when the pipette (or other fluid applicator) is retracted from the well. This will be further described in section 2 below.
Method 2- -fluid input suitable for subsequent heating
The above described method is suitable for room temperature operation of the device, but may result in air bubbles being present in the
In an alternative method, the user proceeds in exactly the same manner as in
In other embodiments of the method, the array elements of the device may be controlled to further ensure that working fluid loaded into the device is not inadvertently withdrawn when the pipette is retracted. This will be further described in section 2 below.
Method 3-fluid extraction
The pipette ports described in this application are bidirectional: they can be used for extracting liquids as well as for injecting fluids. In order to extract the working fluid from the device, it is preferred that the working fluid should be placed as close as possible to the relevant pipette port and that a "shrink-hold" electrode pattern be applied, for example as described in EP 3311919.
Once this adaptive retention pattern is applied, the user should pick up the pipette, push the plunger down to the desired draw volume, insert the pipette into the associated pipette port, and slowly allow the plunger to return. Assuming that the pipette has a sufficiently high aspiration volume, the desired droplet is successfully withdrawn. (the "working fluid" that is removed need not be the same "working fluid" loaded into the fluid chamber, e.g., if an assay is being performed. in this case, to avoid contamination of the fluid being withdrawn, the pipette used for this fluid removal is preferably a different pipette, or has a new disposable pipette tip attached, rather than the one used for fluid loading into the device.)
Although
2. Array element control to assist in loading working fluid
The basic concept of this aspect of the invention is to control the array elements of the EWOD microfluidic device to direct fluid loaded into the fluid channels of the EWOD device to a "safe" position and to provide feedback to the user that this has been done. As a result, all of the loaded working fluid remains on the device (although filler fluid/oil may be lost) when the pipette tip is retracted from the device.
The array element control of this aspect may be applied in conjunction with the fluid loading method described in
Method A-fluid Loading
The simplest example of array element control for assisted loading of working fluid is shown in fig. 6, which shows a top view of an EWOD device having a lower substrate that is more extensive than an upper substrate, thus providing loading holes along one side of the top substrate. It is desirable to load the working fluid into a first region (or target region) of the microfluidic device, such as
In this aspect, the microfluidic device has a plurality of independently addressable array elements (e.g., AM-EWOD microfluidic devices), where each array element corresponds to a respective region of a fluid chamber. As described with reference to fig. 1, the array elements of the microfluidic device may be defined by respective
In the method of fig. 6, a second set of one or more array elements corresponding to a
In this embodiment, it is assumed that the controllable array elements are provided up to the
In fig. 6 (b), the end of a pipette or other fluid applicator is placed adjacent to the
The fluid is then dispensed from a pipette or other fluid applicator. This may be performed, for example, as described above with reference to "
The EWOD control device then deactivates the second set of array elements defining the
Once the fluid moves into the
As shown in fig. 6 (f), the EWOD control device then deactivates the first set of array elements defining a first (target)
As noted, at the end of step (d), feedback is preferably provided to the user so that the user knows that fluid has moved into the
There are many variations on this simplest case. First, there are variations of the array element actuation pattern applied, and these will be described below. Second, each of these different actuation patterns may be applied to different device structures, including:
a) a simple two-substrate device as above, without a housing, where controllable EWOD array elements are provided up to the injection point (as shown in fig. 6);
b) as above, but with a physical gap between the pipette injection point and the nearest controllable EWOD array element (as described with reference to fig. 7);
c) a device having a housing as described in
As shown in fig. 7, the applicability of each actuation pattern to these 3 different device types sometimes depends on the device cell gap, and (in many cases) will depend on using a method of "pushing" the pipette through "stop" to push the fluid away from the end of the pipette using a temporary bubble and onto one or more element electrodes of the EWOD device. Some steps of the method of fig. 7 are similar to corresponding steps of the method of fig. 6, and only the differences will be described.
In the method of fig. 7, it is assumed that the controllable array elements of the EWOD device are not provided until the
Thus, as shown in fig. 7 (d), the pipette (or other fluid applicator) is further actuated to dispense bubbles of air (or other fluid different from the working fluid being dispensed) so as to fully load the working fluid into the
In a modification of this method, the target area may be sufficiently close to the well 66 so that once the working fluid has been loaded into the microfluidic device, as shown in fig. 7 (d), the working fluid may be moved directly to the target area by actuating groups of array elements defining the
In the method of fig. 7, feedback is again preferably provided to the user at the end of step (d) to let the user know that fluid has moved into the
This aspect is not limited to the particular actuation pattern of fig. 6 or 7, and many variations are possible. For example, the description of these methods assumes that the shape of the
Method B-fluid Loading
In this method, the array elements are not initially actuated, but are actuated upon sensing that working fluid is introduced into the fluid chambers of the device, for example in any of the ways described with reference to method a above. This corresponds to fig. 6 or fig. 7, but the second set of array elements is not actuated until it is detected that the working fluid has entered the fluid chamber.
In a related variation, the array elements are initially not actuated, and are actuated again upon sensing that working fluid is introduced into the fluid chambers of the device. However, in this variation, the groups of array elements that are actuated are time dependent such that the
As described further below, groups of array elements actuated to define the time-dependent
Method C-fluid Loading
In this approach, the array element actuation pattern is not changed at all. Actuating the fixed set of one or more array elements to define the actuation area of the device at a "safe" distance from the edge of the EWOD fluid channel ("safe" meaning that the pipette (or other fluid applicator) can be retracted without removing any working fluid from the EWOD channel if the fluid reaches the actuation area-when it is determined that the fluid has reached the actuation area of the device, a control means, such as the aforementioned EWOD control unit, gives or causes an audible or visual cue to the user to retract the pipette.
This second variant corresponds to the method of fig. 7, with the difference that the one or more array elements of the fixed set are successively actuated until after the pipette has been retracted, so that the
Method D-fluid Loading
This method is a combination of the second and third methods, where the array elements are not initially actuated, but once the fluid has reached the "safe zone" of the EWOD channel (e.g., has reached the target region 70), the time-dependent array element sets are actuated. This variant can be used in the case where there are no electrodes at all within the "unsecured" area of the EWOD channel, and other cases where there are electrodes in this area may be advantageous. The actuated array element groups may be based on sensed working fluid volumes to provide adaptive control of the array element actuation, or may be actuated according to a preset pattern.
The above description of
The size and/or location of the "unsafe" areas may be determined by the device manufacturer/supplier based on characteristics of the microfluidic device such as the cell gap and the size of the
Defining the size and/or location of the "unsafe" areas can be as simple as defining a conservative unsafe zone around each injection point. Once it is determined that the unsafe zone has been successfully traversed by the loaded fluid and the unsafe zone is vacated (possibly for a certain amount of time), a signal may be given that the pipette may be retracted.
One factor that can influence which array element actuation pattern is used is the structure of the microfluidic device, as listed in the table below.
Note that in all cases, all methods a to D should be possible above the critical cell gap of the microfluidic device. This table addresses a case of interest, i.e., devices with lower cell gaps, where fluid loading is more challenging. In the case where there is a physical gap between the end of the pipette (or other fluid applicator) and the applied electrode, an air bubble would be required to separate the fluid from the pipette.
In this table, "yes (bubble)" indicates that the method may be applied, but for devices with low cell gaps, it may be necessary to dispense a bubble to force the dispensed fluid into the fluid chamber of the device.
Note that successful loading of fluid into a device without a plastic housing ((a) and (b)) will be highly dependent on the cell gap of the device, and there will be a critical cell gap below which fluid loading would not be possible without a housing that can be sealed around the fluid applicator. For the case (b) where the electrode is not adjacent to the pipette, it is expected that the critical cell gap will be higher. The exact cell gap will depend on the particular filler fluid and working fluid.
After the pipette has been retracted, the droplet can then re-enter the "unsafe" area of the device, since it is no longer unsafe without the pipette. Allowing this may be advantageous as it allows better use of the EWOD channel region for subsequent droplet operations and, therefore, any of the array element actuation patterns described above may be utilized so as to allow the droplets to return to the "unsafe" region once the pipette has been retracted. For example, in the case of manual operation, once the pipette has been retracted, the user may give some signal (e.g. a keystroke or mouse click) to indicate this to the controller, and may then enable the EWOD control unit to actuate the array element to aspirate the droplet into the previous "non-safe" area. Similarly, in a fully robotic embodiment, a control unit controlling the physical position of the pipette or a sensor monitoring the position of the pipette may provide a signal indicating that the pipette has been retracted.
Method E-Loading fluid
This method represents an alternative embodiment of method a described with respect to fig. 6 (a) to (f). In this embodiment, the array elements defining the
This embodiment is illustrated in fig. 8, fig. 8 being a partial plan view of an AM-EWOD device. This figure illustrates a "side loading" embodiment in which the upper substrate of the AM-EWOD device is smaller than the lower substrate, as shown in fig. 5 (a).
The array elements defining the narrow
As fluid is dispensed from the fluid applicator, the working fluid preferentially travels along the activated array elements defining the
Once the introduced working fluid has moved into the
The
Advantageously and preferably, the
This is preferable to reduce the risk of accidental injection of air bubbles and works as follows: by keeping the width of the second region relatively wide (while still providing the gap 74), the back pressure transmitted through the pipette (or other applicator) is maximized (equivalent to allowing "backwashing" of oil through the gap, but this "backwashing" is minimized). Thus, any air bubbles entering the port tend to remain in the region of the port/fluid applicator and be withdrawn into the fluid applicator when the pressure is released (rather than being injected out of the fluid applicator and onto the array or being trapped at the edge of the
Fig. 9 shows another embodiment. This generally corresponds to the embodiment of fig. 8, with the difference that in the embodiment of fig. 9, the
In this embodiment, the triangular shape of the EW pattern is an advantageous feature in maintaining a narrow filler fluid gap in the second region for the reasons described above. One benefit of controlling the
According to a refinement of the above method, an integrated capacitive sensor of an active matrix EWOD device can be used to implement feedback to operate the device in a "closed loop" fashion. Thus, as the working fluid progresses through the narrow second region, the applied actuation pattern may be modified over time in conjunction with the position of the working fluid as determined by the capacitive sensor circuit.
In particular, as shown in (a) to (c) of fig. 10, as the leading edge of the working fluid advances, the width of the actuation pattern may be narrowed thereafter. In these figures, the shaded areas also correspond to the portions of the device occupied by the introduced working fluid. Initially, as shown in fig. 10 (a), the edge 76 of the working fluid is close to the
An advantage of the "closed loop" operation described herein (which uses feedback from a capacitive sensor to determine the position and shape of the advancing working fluid droplet) is that the method is more tolerant of variations in the velocity at which the user uses the fluid applicator to manually introduce the working fluid, resulting in more desirable introduction of the working fluid into the chamber, including the following: more reliable fluid input, avoidance of fluid contact with spacers, avoidance of air bubble injection; all of which are independent of the speed or technology of the user. The same sensor feedback provides improved control over the rate and volume of working fluid delivered to the device when using a smart fluid applicator.
During loading of the working fluid into the chamber, status information/notifications may be provided to the user. Initially, the user may receive a notification indicating successful start of the loading process based on feedback from the capacitive sensor, confirming that the working fluid has been initially contacted with the
If an automated fluid applicator is used, sensor feedback may be used to control the rate of fluid dispensing and the volume of fluid dispensed.
In principle, the embodiments of fig. 10 (a) to (c) may be implemented without sensing the position of the fluid edge, for example by an EWOD control device applying a time-varying EWOD element actuation pattern in a preprogrammed manner.
Because the
Although fig. 9 shows the
Method F-Loading fluid
In a modification of "method E" described above, a droplet separation operation is performed in the
Advantages of this embodiment may include one or more of the following:
(1) creating a reservoir of small volume of working fluid that may be less than the minimum volume of working fluid that may be dispensed by the fluid applicator. Typically, the volume treated by the fluid applicator is at least 2 microliters or more. However, for many EWOD applications, it is generally preferred to perform microfluidic manipulation, typically nanoliter-scale, of fluids having volumes significantly less than 2 microliters. The benefits of this ability to apply such small volumes of working fluid are: minimizing the use of expensive or precious samples/reagents; the use of a minimum volume of working fluid also has the benefits of: effectively using the fluid treatment area on the electrowetting array.
(2) A reservoir for generating a precise volume of working fluid based on capacitive sensor feedback. This embodiment enables the volume of the reservoir produced to be controlled to an accuracy of a few percent, typically more accurate than the volume dispensed by a pipette. As the user introduces working fluid from the fluid applicator, the sensor feedback can be used to control the size of the working fluid zone, and thus the volume of working fluid transferred to the
Method G-fluid extraction
Embodiments of the present disclosure have been described above with reference to loading a working fluid into an AM-EWOD device. The present disclosure may also provide a method of extracting a working fluid from an EWOD device, such as an AM-EWOD device.
For example, after running a reaction protocol in an EWOD or AM-EWOD device, a first region 80 of the device will contain the resulting working fluid, and it may be desirable to draw some or all of the working fluid from the EWOD device in region 80 for analysis. Another benefit of using a narrow second region is that during the process of extracting such working fluid, the working fluid is first directed to the first (target) region 80 before being directed along the narrow second region 82 towards the
In some embodiments, the fluid applicator may be introduced into the port adjacent to the
The method of extracting working fluid from the array may follow a similar procedure to the fluid loading method described above, but operate in the reverse order.
Essentially, the EWOD control device actuates an element in a second region 82, the second region 82 extending fully or partially between a first region 80 of the device and the
The beneficial advantages of the improved method of extracting a sample from an EWOD device are described below with reference to fig. 11 and 12. In these figures, the shaded areas correspond to the parts of the device occupied by the working fluid, which is desired to be at least partially extracted. As in the loading method described above, the width of the working fluid region at its closest point to the orifice is less than the width of the orifice. In fig. 11, the region of working fluid extends to and through the aperture such that the point of the second region 82 closest to the aperture is located at the aperture, but in other embodiments the second region may not extend to the aperture. The smaller width of the working fluid region results in a gap 84 being provided between the working fluid and the edge of the orifice. Advantageously and preferably, the gaps 84 are small and can be controlled (by selecting the actuation pattern) to have, for example, a width of one or two array elements per gap 84.
As noted above, the beneficial aspects of this process are achieved by: defined activation of regions of the electrowetting array is performed to accurately position the working fluid relative to the port through which the working fluid is withdrawn. Generally, the first region 80 (i.e., the portion of the fluid chamber containing the working fluid to be extracted-which may also be referred to as a "reservoir") may be further from the
One reason for the increased distance from the reservoir 80 to the spacer is: initially inserting the end of the fluid applicator into the port may transfer mechanical force through the filler fluid, which may cause the working fluid in the reservoir to momentarily become slightly displaced or agitated. It is particularly desirable to ensure that any such oscillation does not contact the working fluid in the reservoir 80 with the spacers. Thus, locating the working fluid in the first region/reservoir 80 further away from the spacer than the
The use of a narrower second region 82 when withdrawing working fluid from the chamber is to facilitate the movement of filler fluid out of the port region back into the chamber, wherein the filler fluid present in the port region is replaced by the working fluid being withdrawn. In some cases, it is preferred that the gap 84 between the edge of the spacer and the second region 82 be wider than the
Thus, when a fluid applicator that has been inserted into a port begins to draw working fluid, there is a reduced tendency for any filler fluid to be co-drawn with the working fluid. The result is a smaller volume of filler fluid that is pumped with the working fluid. This has a number of benefits, in particular it can minimise the negative impact that the filler fluid may have on subsequent processing that the working fluid may undergo. It is possible that by reducing the volume of the extracted filler fluid, a subsequent cleanup step may not be required to remove excess filler fluid from the extracted working fluid.
Thus, as described above, the advantages of the capacitive sensor function for detecting fluid position facilitate closed loop feedback in device operation, allowing real-time modification of the actuation pattern applied to the electrowetting array according to the position and shape of the working fluid droplets.
Thus, after the end of the fluid applicator has been inserted into the port, the EWOD control device actuates the EWOD element in the second region 82, which second region 82 extends from the first region 80 toward the
Dashed line 80a in fig. 11 represents the boundary between reservoir/first region 80 and second region 82. As explained above, this boundary may be considered a conceptual boundary and is defined by the actuation of the array elements.
The shape of the second region 82 in fig. 11 generally corresponds to the shape of the
In the fluid loading embodiments of fig. 10 (a) to (c), the shape of the
The shape of the second region 82 in which the EWOD element is actuated changes over time as the working fluid to be extracted moves towards the orifice. It can be seen that as the working fluid moves towards the aperture, the length of the extraction region 82a decreases, while the length of the transition region 82b increases and widens to have a width equal to the width of the reservoir region. As noted above, the EWOD control device can deactivate the EWOD element in the reservoir region 80 upon activation of the EWOD element in the second region 82. In this method, the movement of the fluid is controlled by the changing contact angle at the leading fluid edge 86. As the length of the extraction region 82a decreases and the transition region 82b moves toward the aperture, this movement of the transition region 82b will substantially urge working fluid into the extraction portion 82a from which the negative pressure of the fluid applicator can draw fluid out of the chamber of the device. Thus, all array elements in the second region 82 may remain actuated during fluid extraction; alternatively, the array elements behind the fluid trailing edge 88 may be in a non-actuated state as the fluid trailing edge 88 moves toward the aperture.
As with the described fluid loading embodiments, the EWOD control device may control actuation of elements of the EWOD device based on received position information about the working fluid in the device or according to a preprogrammed control scheme.
Selective activation and deactivation of the array elements near the
Method H-extraction Process
Another embodiment of the extraction process is similar to method G, but here a droplet separation operation may be performed to separate the droplets in the second region 82 into two discrete droplets, as shown in fig. 13. Thus, in droplet extraction, a
The invention described with reference to methods a to F relates to the safe loading of the complete volume of working fluid present in the fluid applicator, eliminating or at least significantly reducing the risk of working fluid being erroneously withdrawn from the working area of the microfluidic device when the pipette is retracted, or ensuring that the working fluid is extracted while eliminating or at least significantly reducing the risk of filler fluid being erroneously extracted with working fluid.
Although many measures may be taken to prevent the working fluid from being withdrawn incorrectly during fluid loading (as described above), it should be possible to detect whether this has occurred by using a sensor array integrated into the EWOD electrode array. If, for example, the assay protocol requires loading 5 microliters of working fluid, but the sensor array records that only 3 microliters have been loaded by some user incident (such as the working fluid being loaded is incorrect in volume, or the correct volume of working fluid was initially loaded but some of the working fluid was inadvertently withdrawn upon withdrawal of the fluid applicator), a warning can be issued to the user to add more fluid, try again or withdraw 3 microliters, and repeat.
Similarly, the correct volume may in fact have been successfully loaded, but the fluid within the microfluidic device is incorrectly positioned (which will depend on the type of software function selected), or it may have merged with a nearby droplet, which may have been loaded from a nearby (or the same) fluid loading well. Also, a sensor array built into the device can be utilized to alert the user that such an event has occurred and prompt them to take appropriate action (e.g., remove the cartridge from the experiment and then start over).
Another possibility is that a droplet of working fluid is finished in the correct position, but in the process of reaching the correct position, the droplet may be temporarily located in an unintended area of the device. This is likely to occur if the user pushes the pipette plunger very forcefully through the stop of the pipette and the air bubble injected is much larger than the minimum required to nudge the dispensed working fluid onto the desired electrode. Even if the mis-positioning is only brief, this may lead to contamination problems in the case of assays where the area of the EWOD array is intended to remain pristine and unused prior to the introduction of a particular type of working fluid, for example, where multiple samples are to be analyzed independently within the same device. Again, real-time sensor information may be used to alert the user of any such risk, thus allowing the user to decide whether to continue or resume again.
All embodiments described herein may alternatively be implemented by using an electronic pipette controlled by or in conjunction with an EWOD control unit that controls actuation of an array element of an EWOD microfluidic device. Such pipettes can be automated to provide the correct loading speed for the stage of loading the working fluid and can control the additional "push through" stop "stage very precisely to avoid potential user error.
In the case of manual fluid loading, a warning or alarm (or other output) is provided to the user and may be, for example, an audible and/or visual output, while in the case of automatic or robotic fluid loading, the output is provided to a control unit that controls the automatic or robotic fluid loading, such as an EWOD control unit, and may be, for example, an electrical or optical signal.
For example, it would be advantageous to control the rate of formation of the air bubble to prevent the user from pushing too forcefully through the "stop" so that the air bubble separates from the pipette tip. If the air bubble should detach from the pipette tip, this would mean that the air bubble is not recoverable before the pipette is retracted. Automatic pipettes can prevent such accidents.
It would also be advantageous to control the amount of air injected so that the air bubbles are just large enough to allow the fluid to contact the electrodes. Sensor feedback from the EWOD array element will provide information (possibly wireless) to the pipette to control this phase of fluid injection. Once the fluid is placed on the electrode, it is safe to begin retracting the air bubbles and excess filler fluid during the time it takes for the droplet to reach the safe zone, as shown in fig. 7 (d). This will speed up the fluid loading process.
In addition, such intelligent pipettes may also be advantageous because they may be programmed to follow the complete loading sequence of a particular assay or protocol to be performed on the device. It can automatically pump the correct volume for the various ports. All the user needs to do is change pipette (or remove disposable pipette tip from pipette and replace it with a clean pipette tip), submerge the new pipette/new pipette tip into the correct reagent tube and dock with the correct port.
A security feature may also be built, i.e. to detect if the user has selected the correct port. If not, the pipette will automatically retract the droplet to the tip, and the software will remind the user which port should be loaded and retry immediately.
The pipette may also assist in drawing fluid: the speed of the pumping may be adapted to the contracted volume of the droplet sensed on the device to minimize user error.
Some of the above embodiments relate to dispensing air bubbles from a pipette to force the dispensed fluid into a fluid channel of a microfluidic device. Some users may feel uncomfortable with the concept of injecting air bubbles (albeit temporarily) into their devices. If this is the case, the user may instead load the fluid applicator with both the filler fluid and the working fluid, such that the fluid dispensed after dispensing the working fluid and following the working fluid onto the device is the filler fluid, rather than air. The way in which the oil (or other filler fluid) is dispensed behind the working fluid can be exactly the same way in which air bubbles are dispensed, but with the advantage that the user is not surprised by seeing the bubbles on the device.
With a manual pipette, dispensing oil (or other filler fluid) after the working fluid may be achieved, but may be difficult to perform. However, if programmed correctly, a smart pipette (as described above) can easily perform dual fluid loading.
The invention as described with reference to methods G and H is directed to ensuring that working fluid is extracted while eliminating or at least significantly reducing the risk of filler fluid being extracted incorrectly with the working fluid instrument.
Some of the above embodiments relate to sensing the presence and/or location of fluid within an EWOD microfluidic device, for example sensing that fluid has reached a
(i) the element electrodes are selectively actuated by supplying voltages to the array element electrodes. Thus, any liquid droplet present at the array element 51 may be actuated or de-actuated by the electrowetting effect.
(ii) The presence of a liquid droplet at the location of the array element is sensed. The means of sensing may be capacitive, optical, thermal or some other means. Capacitive sensing can be conveniently and efficiently employed using impedance sensor circuits as part of the array element circuitry.
Exemplary configurations of array element circuits including impedance sensor circuits are known in the art and are described in detail, for example, in US8653832 and commonly assigned uk application GB1500261.1, both of which are incorporated herein by reference. These patent documents include descriptions of: how the droplet can be actuated (by electrowetting) and how the droplet can be sensed by capacitive or impedance sensing means. In general, capacitive and impedance sensing may be analog, and may be performed simultaneously or nearly simultaneously at each element in the array. By processing the return information from such sensors, the control system can determine the location, size, centroid and perimeter of each liquid droplet present in the microfluidic device in real time or near real time.
Alternatively, an external sensor module may be provided for sensing the droplet properties. For example, optical sensors known in the art may be used as external sensors for sensing properties of the droplets. Suitable optical sensors include camera devices, photosensors, charge-coupled devices (CCDs), image-similar image sensors (image sensor), and the like.
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