Patent Application
20220326137
The present invention relates to a microfluidic device, and to a method of manipulating fluid droplets in such a device. More particularly, the invention relates to an Electro-wetting on Dielectric (EWOD) microfluidic device such as, for example, an Active Matrix Electro-wetting on Dielectric (AM-EWOD) microfluidic device. Electro-wetting-On-Dielectric (EWOD) is a known technique for manipulating droplets of fluid on an array. Active Matrix EWOD (AM-EWOD) refers to implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs).
Microfluidics is a rapidly expanding field concerned with the manipulation and precise control of fluids on a small scale, often dealing with sub-microlitre volumes. There is growing interest in its application to chemical or biochemical assays and synthesis, both in research and production, and applied to healthcare diagnostics (“lab-on-a-chip”). In the latter case, the small nature of such devices allows rapid testing at point of need using much smaller clinical sample volumes than for traditional lab-based testing.
A microfluidic device can be identified by the fact that it has one or more channels (or more generally gaps) with 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 a variety of interesting measurements including molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients and enzyme reaction kinetics. Other applications for microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, enzymatic assays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning and chemical gradient formation. Many of these applications have utility for clinical diagnostics.
Many techniques are known for the manipulation of fluids on the sub-millimetre scale, characterised principally by laminar flow and dominance of surface forces over bulk forces. Most fall into the category of continuous flow systems, often employing cumbersome external pipework and pumps. Systems employing discrete droplets instead have the advantage of greater flexibility of function.
Electro-wetting on dielectric (EWOD) is a well-known technique for manipulating discrete droplets of fluid by application of an electric field. It is thus a candidate technology for microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in “Digital microfluidics: is a true lab-on-a-chip possible?” (R. B. Fair, Microfluid Nanofluid (2007) 3:245-281).
On the top substrate 36 is a second hydrophobic coating 26 with which the liquid droplet 4 may come into contact. Interposed between the top substrate 36 and the second hydrophobic coating 26 is a reference electrode 28.
The droplets have a contact angle 6 with the hydrophobic surface of the insulator layer. The contact angle 6 is determined by the balancing of the surface tension components (1) from the hydrophobic surface to the liquid of the droplets (γSL) interface, (2) from the liquid of the droplets to the surrounding fluid medium (γLG) interface, and (3) from the hydrophobic surface to the surrounding fluid medium (γSG) interface. Where no voltages are applied, the contact angle 6 satisfies Young's law, and is of size θ given by the equation:
cos θ=((γSG−γSL)/γLG) (Equation 1).
In operation, voltages termed the EW drive voltages, (e.g. VT, V0 and V00 in
As described above with respect to the representative EWOD structure, the EWOD channel or gap defined by the two substrates initially is filled with the nonpolar filler fluid (eg oil). The liquid droplets 4 including a polar material, i.e., the droplets to be manipulated by operation of the EWOD device, must be inputted from an external “reservoir” of fluid into the EWOD channel or gap. The external reservoir may for example be a pipette, or may be a structure incorporated into the plastic housing of the device. As the fluid from the reservoir for the droplets is inputted, filler fluid gets displaced and is removed from the EWOD channel.
Example configurations and operation of EWOD devices are described in the following. U.S. Pat. No. 6,911,132 (Pamula et al., issued Jun. 28, 2005) discloses a two dimensional EWOD array to control the position and movement of droplets in two dimensions. U.S. Pat. No. 6,565,727 (Shenderov, issued May 20, 2003) further discloses methods for other droplet operations including the splitting and merging of droplets, and the mixing together of droplets of different materials. U.S. Pat. No. 7,163,612 (Sterling et al., issued Jan. 16, 2007) describes how TFT based thin film electronics may be used to control the addressing of voltage pulses to an EWOD array by using circuit arrangements very similar to those employed in AM display technologies.
EWOD devices are useful for miniaturising and automating biochemical/chemical processes through the manipulation of aqueous droplets in an oil matrix; EWOD devices may be used to split, move, merge and mix droplets. Typically biochemical/chemical workflows involve carrying out a sequence of reactions on biological/chemical samples, with each step involving the merging of one or more droplets containing the reagents necessary for that step with the sample droplet. The process of merging of droplets containing reagents into the sample droplet means that the volume of the sample droplet typically increases as the workflow proceeds, and the reaction droplet's composition becomes the sum of the work flow product(s) and any remaining reactants and by-products. Due to the enclosed nature of EWOD devices the reaction droplet cannot easily be concentrated by conventional concentration techniques such as solvent evaporation. Also, it may be desired to extract one or more components from the reaction droplet, but the enclosed nature of EWOD devices again means that components of the reaction droplet cannot be easily selected and then separated into a second droplet.
Magnets and biomolecule targeting magnetic beads may be used to bind and remove components of a droplet in an EWOD device, but this process may be inefficient and poorly selective.
The EWOD device is typically operated as part of a microfluidic system comprising a control system configured to control actuation voltages applied to the electrode array of the EWOD device (or other microfluidic device) to perform manipulation operations to the fluid droplets. For example,
The control electronics 3a may further receive data signals 9 from one or more sensors (not shown) of the EWOD device 2. The sensor data signals 9 may include signals obtained by operating the EWOD array elements in a sensing mode, and/or may include signals obtained by one or more sensors external to the EWOD array elements such as some or all of illumination and/or detection optics, a thermal control unit or magnetic systems for interacting with the droplets 4.
Active Matrix EWOD (AM-EWOD) devices typically include a droplet sensor incorporated into the bottom substrate, for example as disclosed in US application 2012/0007608 (Hadwen et al., published Jan. 12, 2012), which describes how an impedance (capacitance) sensing function can be incorporated into some or all of the array elements of the array. The impedance sensor incorporated into an array element may be used for determining the presence and size of a liquid droplet present in that array element.
A first aspect of the present disclosure provides a method of manipulating droplets in an electro-wetting on dielectric (EWOD) device, the EWOD device having first and second substrates defining a fluid chamber therebetween and a plurality of electro-wetting electrodes, the method comprising selectively actuating the electro-wetting electrodes to: cause first and second droplets in a fluid medium in the fluid chamber of the EWOD device to contact each other to form a droplet interface bilayer, the first droplet containing fluid of a first composition including a first solvent species and the second droplet containing fluid of a second composition, the second composition being different to the first composition and containing a higher osmolarity solute, than the first composition and so having a higher osmotic pressure; maintain the first and second droplets contacting each other to maintain the droplet interface bilayer and thereby allow a solvent species to pass from the first droplet to the second droplet via the droplet interface bilayer; and cause the first droplet to separate from the second droplet. (The “osmolarity” of a solution is a measure of the solute particle concentration, and is explained further below.)
A method of this aspect results in transfer of solvent from the first droplet to the second droplet, whereby the fluid in the first droplet becomes more concentrated and the fluid in the second droplet becomes more diluted. This provides a convenient way of altering the concentration in a fluid droplet within an EWOD device. For example, the first droplet may be a reaction droplet that has been obtained via a sequence of reaction steps involving merging of droplets, so that the reaction droplet's composition has become the sum of the work flow product(s) and any remaining reactants and by-products. The invention allows the reaction droplet to be concentrated before the droplet undergoes further reaction steps.
A method of this aspect also results in a reduction in size of the first droplet by the transfer of solvent from the first droplet to the second droplet, and an increase in the size of the second droplet. A method of this aspect may alternatively be used to adjust the size of a droplet within an EWOD device, for example to reduce the size of a droplet (for example to increase its speed of movement within the EWOD device, or to prepare the droplet for removal from the EWOD device), or otherwise to adjust the size of a droplet to be equal to a desired size for any desired reason. This provides a way to make droplets of sizes that would otherwise not be possible for a given EWOD device geometry. For the successful splitting off of a daughter droplet from a main droplet, the daughter droplet has a minimum droplet diameter about 3×the cell gap height—i.e. as defined by the spacer 32 in
The first droplet may contain a first solute at a first osmolarity in a first solvent and the second droplet contains a second solute at a second osmolarity in the first solvent, and the second osmolarity may be higher than the first osmolarity whereby first solvent species passes from the first droplet to the second droplet via the DIB.
The method may comprise maintaining the two droplets contacting each other so as to maintain the droplet interface bilayer for a sufficient time to allow the osmotic pressure of the solution in the first droplet to become equal or approximately equal to the osmotic pressure of the solution in the second droplet.
The method may comprise, after actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet, further selectively actuating the electro-wetting electrodes to: cause the first droplet to contact a third droplet to form a droplet interface bilayer, the third droplet containing fluid of a third composition including the first solvent species, the third droplet having an osmotic pressure lower than the osmotic pressure of the first droplet after the first droplet has been caused to separate from the second droplet; maintain the first and third droplets contacting each other to maintain the droplet interface bilayer and thereby allow the first solvent species to pass from the third droplet to the first droplet via the DIB; and cause the first droplet to separate from the third droplet.
The method may comprise selectively actuating the electro-wetting electrodes to, before causing the first and second droplets to contact each other to form the droplet interface bilayer: cause a fourth droplet to merge with a fifth droplet to form the first droplet. When the fourth and fifth constituent droplets are merged it is possible that the resultant droplet will not have a uniform fluid composition over its volume, and this may be undesirable. The constituents of the first droplet (the merged droplet) may be mixed to give a more uniform composition by using a method of this aspect to extract solvent from the first droplet, and then re-introducing solvent into the first droplet (for example by bringing the first droplet into contact with a further droplet having a low total solute osmolarity such that solvent passes from the further droplet into the first droplet).
The method may comprise, after actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet, further selectively actuating the electro-wetting electrodes to: cause the first droplet to contact a third droplet to form a droplet interface bilayer, the third droplet containing fluid of a third composition including a second solvent species different from the first solvent species, the third droplet having an osmotic pressure lower than the osmotic pressure of the first droplet after the first droplet has been caused to separate from the second droplet; maintain the first and third droplets contacting each other to maintain the droplet interface bilayer and thereby allow the second solvent species to pass from the third droplet to the first droplet via the DIB; and cause the first droplet to separate from the third droplet.
The method may comprise, after actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet, further selectively actuating the electro-wetting electrodes to: cause the first droplet to contact a third droplet to form a droplet interface bilayer, the third droplet comprising fluid of a third composition, the third droplet having a higher osmotic pressure than the osmotic pressure of the first droplet after the first droplet has been caused to separate from the second droplet; maintain the first and third droplets contacting each other to maintain the droplet interface bilayer and thereby allow the first solvent species to pass from the first droplet to the third droplet via the DIB; and cause the first droplet to separate from the third droplet.
The method may further comprise monitoring the size of the first droplet and/or monitoring the size of the second droplet using a sensor.
The sensor may be comprised within the EWOD device.
The sensor may be an impedance sensor and may be integrated into one of the substrates of the EWOD device.
Selectively actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet may be based on an output from the sensor.
The method may further comprise selectively actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet when one of the first droplet or second droplet has a diameter of less than ×3 the cell gap.
The method may further comprise selectively actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet when one of the first droplet or second droplet has a diameter of between ×1 and ×1.5 of the size of the electro-wetting electrodes.
The method may further comprise monitoring the size of the first droplet and/or monitoring the size of the second droplet while selectively actuating the electro-wetting electrodes to maintain the first and second droplets contacting each other to maintain the droplet interface bilayer; and obtaining information about the first composition relative to the second composition from the monitoring of the size of the first droplet and/or the size of the second droplet.
The second composition may be a known composition.
The method may comprise, while actuating the electro-wetting electrodes to cause the first and second droplets to contact each other to form the droplet interface bilayer, further actuating the electro-wetting electrodes to: cause a fourth droplet to contact the first droplet to form a second droplet interface bilayer, the fourth droplet containing fluid of a fourth composition different to the first composition, the fourth composition having a higher osmotic pressure than the first composition; and maintain the first and fourth droplets contacting each other to maintain the second droplet interface bilayer and thereby allow the first solvent species to pass from the first droplet to the fourth droplet via the second DIB.
The method may comprise, while actuating the electro-wetting electrodes to cause the first and second droplets to contact each other to form the droplet interface bilayer, further actuating the electro-wetting electrodes to: cause a fourth droplet to contact the second droplet to form a second droplet interface bilayer, the fourth droplet containing fluid of a fourth composition different to the second composition, the fourth composition having a higher osmotic pressure than the second composition; and maintain the second and fourth droplets contacting each other to maintain the second droplet interface bilayer and thereby allow the first solvent species to pass from the first droplet to the second droplet via the DIB and to pass to the fourth droplet via the second DIB.
A transporter species for promoting passage through the droplet interface bilayer of the solvent species may be provided in the first droplet and/or in the second droplet
The transporter species may be an aquaporin.
WO2018/067878 provides disclosure of both biological pores and solid state pores. The person skilled in the art would recognise that such species may be utilised as nanopores in the droplet interface bilayers of the present invention.
A second aspect of the disclosure provides a method of manipulating droplets in an electro-wetting on dielectric (EWOD) device, the EWOD device having first and second substrates defining a fluid chamber therebetween and a plurality of electro-wetting electrodes, the method comprising: selectively actuating the electro-wetting electrodes to: cause first and second droplets in a fluid medium in the fluid chamber of the EWOD device to contact each other to form a droplet interface bilayer, the first droplet containing fluid of a first composition including a first solute species and the second droplet containing fluid of a second composition different to the first composition, maintain the first and second droplets contacting each other to maintain the droplet interface bilayer and thereby allow the first solute species to pass from the first droplet to the second droplet via the DIB; and cause the first droplet to separate from the second droplet.
A method of this aspect results in transfer of solute from the first droplet to the second droplet. This provides a convenient way of altering the concentration of a particular component or components in a fluid droplet within an EWOD device. For example, the first droplet may be a reaction droplet that has been obtained via a sequence of reaction steps involving merging of droplets, so that the reaction droplet's composition has become the sum of the work flow product(s) and any remaining reactants and by-products. This aspect allows, for example, an undesired solute species to be extracted from the reaction droplet or the undesired solute species to be diluted in the reaction droplet before the droplet undergoes further reaction steps, allows the reaction droplet to be provided/replenished with a desired solute species before the droplet undergoes further reaction steps, and/or allows a solute species to be extracted from the reaction droplet for analysis or to undergo further reaction steps.
A transporter species for promoting passage through the droplet interface bilayer of the first solute species may be provided in the first droplet and/or in the second droplet. When the droplet interface bilayer is formed the transporter species is incorporated into the DIB, and allows the first solute species to pass through the DIB while selectively preventing one or more other species from passing (for example if the first solute species is a relatively small molecule, transport of the first solute species may be facilitated by a transmembrane species that allows smaller molecules to pass while selectively preventing a larger molecule from being transported). One example of a suitable transporter species is a transmembrane transporter protein.
The first droplet may further contain a second solute species and the transporter species may be arranged to block transfer via the droplet interface bilayer of the second solute species.
The method may comprise actuating the electro-wetting electrodes to maintain the droplet interface bilayer between the first droplet and the second droplet for a sufficient time to allow the solute concentration of the first droplet to become equalised between the first droplet and the second droplet.
The method may comprise monitoring the size of the first droplet and/or the size of the second droplet using a sensor.
The sensor may be comprised within the EWOD device.
The sensor may be an impedance sensor and may be integrated into one of the substrates of the EWOD device.
The method may comprise monitoring, with a sensor, the concentration of the solute in one or both of the first and second droplets
The sensor may configured to perform one or more optical measurements on the droplet, and optionally the one or more optical measurements may be selected from fluorescence, absorption, luminescence or optical imaging.
The sensor be configured to measure one or more non-optical properties of the droplet, and optionally wherein the one or more non-optical properties may be selected from conductivity, viscosity, electrical permittivity or pH.
Selectively actuating the electro-wetting electrodes to cause the first droplet to separate from the second droplet may be based on an output from the sensor.
The method may comprise, while actuating the electro-wetting electrodes to maintain the first and second droplets contacting each other to maintain the droplet interface bilayer, promoting transfer of the first solute species from the first droplet to the second droplet.
The method may comprise selectively actuating the electro-wetting electrodes to promote transfer of the first solute species from the first droplet to the second droplet.
The method may comprise inducing an electrochemical or chemical gradient across the droplet interface bilayer to promote transfer of the first solute species from the first droplet to the second droplet.
The method may comprise thermally or optically promoting transfer of the first solute species from the first droplet to the second droplet
The method may comprise providing a magnetic field gradient across the droplet interface bilayer to promote transfer of the first solute species from the first droplet to the second droplet
The second droplet may contain fluid of a second composition including a third solute species, and a second transporter species for promoting passage through the DIB of molecules of the third solute species may be provided in the first droplet and/or in the second droplet thereby enabling the third solute species to pass from the second droplet to the first droplet via the droplet interface bilayer.
The method may comprise, while actuating the electro-wetting electrodes to cause the first and second droplets to contact each other to form the droplet interface bilayer, further actuating the electro-wetting electrodes to: cause a third droplet to contact the first droplet to form a second droplet interface bilayer, the third droplet containing fluid of a third composition different to the first composition, the third composition having the same osmotic pressure as the first composition, wherein a second transporter species for promoting passage through the second droplet interface bilayer of a fourth solute species is provided in the first droplet and/or in the third droplet, and maintain the first and third droplets contacting each other to maintain the second droplet interface bilayer and thereby allow the fourth solute species to pass from the first droplet to the third droplet via the droplet interface bilayer. In this embodiment the fourth solute species may be the same as the first solute species, although this embodiment is not limited to this.
The method may comprise, while actuating the electro-wetting electrodes to cause the first and second droplets to contact each other to form a droplet interface bilayer, further selectively actuating the electro-wetting electrodes to: cause a third droplet to contact the second droplet to form a second droplet interface bilayer, the third droplet containing fluid of a third composition different to the first composition, the third composition having the same osmotic pressure as the first composition, wherein a transporter species for promoting passage through the second DIB of the first solute species is provided in the second droplet and/or in the third droplet, and maintain the second and third droplets contacting each other to maintain the second droplet interface bilayer and thereby allow the first solute species to pass from the first droplet to the second droplet via the droplet interface bilayer and from the second droplet to the third droplet via the second droplet interface bilayer.
The method may comprise further selectively actuating the electro-wetting electrodes to: retrieve the second droplet.
A third aspect of the disclosure provides an electro-wetting on dielectric (EWOD) device, the EWOD device having first and second substrates defining a fluid chamber therebetween, a plurality of electro-wetting electrodes, and an electrode control unit for selectively actuating the electro-wetting electrodes to perform a method of the first aspect or of the second aspect.
A fourth aspect of the disclosure provides a microfluidic system comprising: an electro-wetting on dielectric (EWOD) device, the EWOD device having first and second substrates defining a fluid chamber therebetween, and a plurality of electro-wetting electrodes; an electrode control unit for selectively actuating the electro-wetting electrodes to perform a method of the first aspect or of the second aspect; and a processor for controlling the electrode control unit to generate actuation data signals for provision to the electro-wetting electrodes of the EWOD device.
In the third or fourth aspect, the EWOD device may be an active matrix EWOD device.
Preferred embodiments of the present invention will now be described by way of illustrative examples with reference to the accompanying figures, in which:
Initially, two fluid droplets, Droplet A, 4A, and Droplet B, 4B are present in the fluid space of the EWOD device, as shown by the pair of droplets in
As used herein, specifying that a first droplet and a second droplet “may be manipulated so as to be brought together by appropriate actuation of the array element electrodes of the EWOD device” cover a case where array element electrodes of the EWOD device are actuated to hold the first droplet stationary and move the second droplet into contact with the first droplet, a case where array element electrodes of the EWOD device are actuated to hold the second droplet stationary and move the first droplet into contact with the second droplet, and a case where array element electrodes of the EWOD device are actuated to move the first droplet and to move the second droplet so that the first and second droplets make contact with one another.
Droplet A contains fluid (eg liquid) of a first composition that includes at least a first solute species in a first solvent. In the case of polar fluid droplets the first solvent is typically water although it could be another polar solvent such as methanol. The second droplet, droplet B, contains fluid (eg liquid) of a second composition that is different to the composition of the fluid of droplet A. The fluid in droplet B contains one or more solute species in a solvent, and the fluid of droplet B has a higher overall solute osmolarity than the fluid of droplet A.
As used herein, a “solute” is a substance that is dissolved in another substance, known as a “solvent” to form a mixture known as a “solution”. As would be understood by someone skilled in the art, a substance may be made up of atoms, molecules or ions, therefore a solute may be molecular, ionic or polymeric. The concentration or molarity of a substance or a solute is defined as the number of moles of a substance or solute per litre of solvent, whereas the osmolarity of a solution is a measure of the number of osmoles of solute particles rather than moles of solute. The distinction arises because some substances can dissociate in solution whereas others cannot. For example an ionic solid dissolves in a solvent to generate dissociated solvated ions, and there may not be a one to one relationship between the molarity and the osmolarity of a solution. For example, sodium chloride (NaCl) dissociates into Na+ and Cl— ions. For every 1 mole of NaCl in solution, there are 2 osmoles of solute particles (i.e., a 1 mol/L NaCl solution is a 2 osmol/L NaCl solution). Both sodium and chloride ions affect the osmotic pressure of the solution. In non-ideal solutions solutes may interact with one another thus reducing the osmolarity of a solution. In general the concentration of the solute in the solution is lower than the concentration of the solvent in the solution.
The solvent in droplet B will generally be the same solvent as the first solvent in droplet A, although the invention is in principle not limited to this. As a result, the solution in droplet B has a higher osmotic pressure than droplet A. (In a case where the solvent (second solvent) in droplet B is not the same as the solvent (first solvent) in droplet A, it is possible that the DIB between droplet A and droplet B will be permeable to both the first solvent and the second solvent, so that the two resultant droplets include both the first solvent and the second solvent—if a droplet having methanol as its solvent and a droplet having water as its solvent were to form a droplet interface bilayer, as an example, one would expect that the DIB would be permeable to both water and methanol. After formation of the DIB, each droplet would therefore be likely to contain a mixture of the first and second solvents. The solute composition of the droplets will depend on the permeability of the DIB to the solute(s) present in the initial droplets.)
According to the method of
When the two droplets are manipulated, for example by electrowetting in an EWOD device, so as to come together, the amphiphilic molecules 14 of one droplet interact with the amphiphilic molecules of another droplet so that the amphiphilic monolayers of the two droplets form a droplet interface bilayer 10 as shown in the right view of
The droplet interface bilayer 10 forms a semi-permeable membrane, so that small molecules are able to pass from one droplet, through the droplet interface bilayer, into the second droplet whereas larger molecules cannot easily pass through the droplet interface bilayer 10. Provided that the molecules of the polar solvent in droplet A are small—for example if the solvent is water or methanol—solvent molecules are able to pass through the droplet interface bilayer. The difference in the osmotic pressure of the solutions in droplet A and droplet B results in the movement of solvent molecules from Droplet A across the droplet interface bilayer 10 into Droplet B.
The solute species in Droplet A and Droplet B are typically large molecules, charged molecules or ions, and cannot pass through the droplet interface bilayer or can pass through the DIB but at a much lower rate than the rate at which solvent molecules can pass through the DIB. For example it is likely that a solute species comprising ions or highly charged molecules would not be able to pass through a DIB, whereas dye molecules that are neutral or have a small charge would probably pass through a DIB, but at a low rate. The transport properties of a DIB depend inter alia on the structure of the DIB, and which surfactants have been used in its formation.
As a result, solvent passes from Droplet A to Droplet B across the droplet interface bilayer, as shown schematically in
The rate at which solvent passes from Droplet A to Droplet B across the droplet interface bilayer will depend inter alia on the difference in osmotic pressure between the fluid in Droplet A and the fluid in Droplet B when the DIB forms between Droplet A and Droplet B. Typically the difference in osmotic pressure between the fluid in Droplet A and the fluid in Droplet B is greater than approximately 5 kPa (equivalent to 1 mM sodium chloride solution) at room temperature. Additionally or alternatively, the difference in osmotic pressure between the fluid in Droplet A and the fluid in Droplet B is typically less than 50 MPa (equivalent to 10 M sodium chloride solution) at room temperature.
If the droplet interface bilayer 10 is maintained for a sufficiently long time the process of solvent transfer from Droplet A to Droplet B will continue until the fluid in Droplet A has the same total solute osmolarity as the fluid in Droplet B, and there is no longer an osmotic pressure difference between the solutions in the droplets making up the droplet interface bilayer. Typically it takes of the order of a few hours for the solute osmolarity in the two droplets to become equal.
The resultant droplets—now referred to as Droplet A′ and Droplet B′ in recognition of their changed fluid compositions—may then be manipulated by electrowetting to separate them from one another as shown in
The method of
In a practical application of the method of
Once Droplet A′ and Droplet B′ have been separated from one another, each droplet may then be separately manipulated as desired. In particular, the modified target droplet may be subjected to further reactions, by merging the droplet with one or more further droplets containing the reagent or reagents necessary for a further reaction step. Additionally or alternatively, the ‘unwanted’ droplet produced by the method of
Further details of the formation of a droplet interface bilayer are given in, for example, Scientific Reports 2016, 6, 39349, 1-9, “In vitro synthesis of Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers” by H Findlay et al. This describes selective transport through DIBs formed at the interface between droplets encased in amphiphilic molecules and the concept of functionalising DIBs with transporters, generating DIB networks and controlling the flow of molecular information in artificial cell systems. Electrowetting as a method of forming and breaking DIBs between droplets is the subject of co-pending UK patent application No. 1721649.0. Electrowetting has been described as a method for making and breaking DIBs, for example in Biomicrofluidics 2012, 012813. Osmosis driven transport across a DIB is proposed as a method of concentrating the content of a droplet and of inducing protein crystallisation in the droplet in Cryst Eng Comm 2012, 14(23), 7865-7868.
In this example Droplet A contains solute molecules of interest 18, and it is desired to extract solvent (for example water) from Droplet A. Droplet B has a higher overall solute osmolarity than the overall solute osmolarity of Droplet A. The solution in droplet B therefore has a higher osmotic pressure than the solution in droplet A, where the osmotic pressure of a solution is the minimum pressure which needs to be applied to a sample of that solution to prevent the flow of its pure solvent across a semipermeable membrane and into the sample, and is a measure of the tendency of a solution to take in its pure solvent by osmosis across a semipermeable membrane. It is not necessary for Droplet B to contain any of the solute molecules of interest 18, as long as Droplet B has a higher overall solute osmolarity than the overall solute osmolarity of Droplet A. Droplet B may, for example, contain a solution of salt (sodium chloride) so that its overall solute osmolarity is higher than that of Droplet A.
Also, Droplet A may contain further solute species in addition to the solute molecules of interest 18, for example salt, again provided that Droplet B has a higher overall solute osmolarity than the overall solute osmolarity of Droplet A.
Once the transfer of solvent from Droplet A to Droplet B is complete, or is complete to a desired degree, the two droplets may then be manipulated by electrowetting to separate the two droplets and break the droplet interface bilayer as shown in
As explained above, the method of
In the method of
A further droplet, Droplet B″, is then manipulated to come into contact with Droplet A′ and form another droplet interface bilayer. Droplet B″ has an overall higher solute osmolarity than the overall solute osmolarity of Droplet A′. As a result, further solvent is extracted from Droplet A′ into Droplet B″, to yield a resultant droplet, Droplet A″, that has an overall solute concentration that is higher than the overall solute concentration of Droplet A′. The process also yields a droplet B′″, that contains solvent extracted from droplet A′. The droplet A″ and droplet B′″ may then be manipulated by electrowetting to separate from one another and break the droplet interface bilayer, and each droplet may then be manipulated separately.
If further reduction of the solvent content of Droplet A″ is required, this can be done by one or more further stages of manipulating Droplet A″ and a yet further Droplet B″″ (not shown) to contact one another, and form a droplet interface bilayer to allow further solvent to flow from Droplet A″ into Droplet B″″.
In the embodiment of
In one implementation of this embodiment Droplet B1 and Droplet B2 have the same initial overall solute osmolarity as one another, which is higher than the overall solute osmolarity of the fluid in Droplet A. Even though there is initially no difference in osmotic pressure between Droplet B1 and Droplet B2, as soon as solvent is transferred from Droplet A to Droplet B1 this reduces the overall solute osmolarity in Droplet B1, thus setting up a difference in osmotic pressure between the contents of Droplet B1 and the contents of Droplet B2 to drive the transfer of solvent from Droplet B1 to Droplet B2.
Further embodiments may combine features of
The methods of
In any of the embodiments of
The methods of
Alternatively, the osmolarity of a particular species could alternatively be measured using a range of methods e.g. electrochemical, optical (absorption and fluorescence) measurements etc. In such cases the new composition of the target droplet may be determined even if the transfer of solvent across the droplet interface bilayer is stopped (by breaking the DIB and separating the droplets) before the overall solute osmolarity of the two droplets have become equal.
If a greater change in the composition of the fluid than can be achieved in one single solvent transfer operation is required, the solvent transfer operation may be repeated (
In addition to removing solvent from the target droplet A, the effect of transferring solvent out of the target droplet A is to reduce its physical size, as shown schematically in
Another application of the invention is therefore to generate a droplet of smaller size than it would otherwise be possible to generate in a particular EWOD device. In general, it is found that, when a droplet is formed by splitting off in an EWOD device, the minimum diameter, parallel to the plane of the fluid gap of the EWOD device, of droplet that can be obtained is approximately three times the thickness of the droplet—that is approximately three times the cell gap thickness between the upper and lower hydrophobic coatings 16, 26 in
The methods of the present invention make it possible to generate a droplet having a diameter, parallel to the plane of the cell gap, that is less than the minimum diameter that would normally be obtained for a given cell gap (that is, a diameter that is less than three times the cell gap thickness)—once a droplet has been provided in the cell gap of an EWOD device, its diameter may be reduced by extracting solvent from the droplet according to any of the methods described herein.
In principle the droplet size may be reduced to any desired size, for example chosen based on the subsequent manipulation/processing intended for the droplet. As example, in one embodiment the droplet diameter may be reduced to be the same or approximately the same as the dimension of an electrode of an array element of the EWOD device in which the droplet is contained. As noted above, the cell gap as defined by the height of the spacer (32) determines the size of a droplet that can be generated in an AM-EWOD device by EWOD forces. Therefore, for smaller array element electrodes (38) the minimum size of a droplet that can be moved by EWOD cannot be generated by EWOD but is accessible by volume reduction via a DIB. Small droplet sizes—and hence small droplet volumes—are particularly useful for applications that involve manipulation of cells and where large numbers of droplets are required. The length or width of an array element could be typically 200 μm, or 100 μm or 50 μm, depending on the design of the device. On actuation of a single array element underneath the droplet, the droplet remains in contact with both the top and bottom substrates of the EWOD device and can therefore be moved by electrowetting.
In principle, the droplet diameter could even be made smaller than the dimension of an electrode of an array element of the EWOD device in which the droplet is contained, but such a droplet could not be manipulated by EWOD manipulation techniques. The minimum size of droplet that can be moved by EWOD forces has a diameter such that the droplet approximately covers a single conductive element electrode (38A, 38B).
In general, the speed with which a fluid droplet can be moved in an EWOD device increases as the size of the droplet decreases. Thus, the limit on the minimum diameter of a droplet that can be obtained in a given EWOD device by conventional EWOD techniques also leads to an upper limit on the possible speed of movement of fluid droplets in that EWOD device. As well as making it possible to concentrate or dilute the fluid composition of a droplet within an EWOD device, the present application therefore, also makes it possible to obtain a greater speed of droplet movement as a result of its ability to decrease the physical size of a droplet.
The present invention may also be used to make small adjustments to the size of a droplet within an EWOD device. Once a droplet has been dispensed into the cell gap of the EWOD device, its size may be adjusted so that the droplet has a desired target diameter—which may be less than or greater than the original diameter of the droplet as dispensed.
In a further embodiment, if one or more array elements of the EWOD device include a droplet sensor, such as an impedance (capacitance) sensor as described in US 2012/0007608, use of the sensor in a feedback mode may be used to allow the creation of droplets of a precise size (and thus also having a precise volume). The sensor may be used to monitor the changing size of the droplets either side of the bilayer, and to determine when the size of one of the droplets reaches a pre-determined target size. The “size” of a droplet may refer to any suitable measure of the droplet, such as a dimension of the droplet as seen in plan view. When the size of the droplet is determined to have reached the pre-determined target size, the droplets may be separated, by using electrowetting actuation to perform a droplet operation that separates the droplets, breaking the droplet interface bilayer, at which point solvent transfer between the droplets ceases.
Additionally or alternatively, in any embodiment in which a sensor is provided for sensing one or more droplet properties, actuation of the electro-wetting electrodes to cause the first droplet to separate from the second droplet may be based on an output from the sensor. For example, as described above control electronics such as the control electronics of
In
Upon a droplet 4C reaching the target size, the droplets may be separated by Electrowetting as shown in
Optionally, a sensor may be used to determine the time dependent volume of droplets 4C and 4D. The sensor measures the volume of the droplet and, because it may do so continuously in real time, is capable of measuring time dependent changes in volume. This is regardless of electro-wetting, and just considering the device as a sensor.
Additionally or alternatively, the sensor can operate simultaneously with an electro-wetting actuation function, performing droplet operations under closed loop feedback. The electrowetting actuation pattern(s) and the timing at which they are applied can be arranged in accordance with the position of the liquid droplet on the array, as detected by the sensor. This feedback mechanism has been shown capable of controlling the volumes of droplets created by splitting or dispensing to a high degree of accuracy and precision, more so than if electrowetting actuation patterns are applied without sensor feedback.
A further application of methods of the invention relates to the detection and/or measurement of properties of a droplet. When a biochemical or chemical workflow is being performed through the manipulation of aqueous droplets in an EWOD device, it is often desired to measure properties of a droplet in order to determine whether or to what extent a reaction has occurred. Examples of droplet measurement/detection techniques include optical measurements of the droplet properties or solute particles within it. Such an optical measurement maybe based for example on absorption, fluorescence, chemiluminescence or the imaging of distinguishable particles, for example cells or beads within the droplets. Alternatively, non-optical properties of the droplet or solute within it may be measured including electrical conductivity of the droplet (which provides a measure of the ionic content of the droplet), pH, electrical permittivity and droplet viscosity. pH may be measured by optical means, for example by determining the change of an optical property of a droplet containing a pH indicator in the solvent, or by electrical means, for example by means of an ion sensitive transistor integrated into the lower substrate. Electrical permittivity and or electrical conductivity may be measured, for example, by an impedance sensor integrated into the lower substrate, as previously described. Viscosity may be measured by determining the speed of movement induced in a droplet for a given actuation voltage. These and other detection techniques may be made more reliable if the fluid composition of the droplet is concentrated using a method of the invention before the measurement is made. For example, in the case of an optical absorption or fluorescence measurement being made on a droplet, the optical absorption or fluorescence signal is expected to be more intense for higher solute concentrations of the droplet.
A further possible application of the invention is in making it possible to stop, or slow, a particular biochemical or chemical reaction occurring in a droplet. For example, some reactions can be slowed or even stopped by increasing the concentration of salt in the fluid in which the reaction is occurring—so extracting solvent from a droplet as described herein may also be used as a way of controlling or reducing the speed of (and possibly stopping) a reaction, by increasing the concentration of salt or other solute species in the droplet.
As set out above, methods of the invention may be used to reduce the volume of a droplet, by extracting solvent from the droplet. A further application of this technique is in reducing the volume of a droplet before it is extracted from the EWOD device, so that the droplet has the correct volume for further measurements to be made on the droplet. This can be more convenient and easier than attempting to reduce the size of a droplet after it has been extracted from the EWOD device.
In a further application of methods of the invention, solvent may initially be extracted from a target droplet according to any of the methods described herein. Solvent may then be re-introduced into the droplet, by bringing the droplet into contact with a droplet having a low total solute osmolarity. This process may be useful in mixing the contents of a droplet that has been formed by merging two or more constituent droplets. If two or more constituent droplets are merged it is possible that the resultant droplet will not have a uniform fluid composition over its volume, and this can lead to inaccuracy in further processing steps on the droplet. The process of removing solvent from the droplet and then re-introducing further solvent will promote the mixing of the droplet constituents to give a more uniform composition.
Such a solvent exchange technique may also be used to change the solvent composition of a droplet. For example, one polar solvent (for example water) may be removed from a droplet, and a different polar solvent (such as methanol) may be introduced into the droplet.
A method of the invention may be used to concentrate the composition of a fluid droplet at any point in a sequence of droplet operations (or “workflow”). For example, it may be used to concentrate an initial sample droplet provided in an EWOD device so as to reduce its size, to allow enough room on the AM-EWOD electrode array to manipulate multiple samples in parallel. The manipulating may include droplet splitting and mixing. Samples loaded onto a device may come from various sources and it may not be convenient or possible for the user to preconcentrate a sample for loading onto the AM EWOD device. Large volume droplets will take up considerable space in the AM EWOD device, and reducing the size of the droplets by a method of the invention may be a convenient way to increase the number of samples that may be processed in parallel in a given EWOD device. The droplets used to concentrate the target droplet(s) could be removed from the device before the workflow is carried out.
Additionally or alternatively, a method of the invention may be used to remove solvent from, or add solvent to, an initial sample droplet in order to generate sample droplets with the correct concentration of a particular component required for a particular workflow. Again this addresses the problem that samples loaded onto a device may come from various sources and have different concentrations.
As a further example, the invention may be applied to the final droplet obtained in a chemical or biochemical workflow, to concentrate the final droplet before extraction of a particular volume of droplet fluid for further analysis.
Another application of methods of the invention is described with reference to
In the first stage of the method of
Optionally, at least one of Droplet A and Droplet B—in this example Droplet A—includes a “transporter” species 30 that will insert, or can be inserted, into the droplet interface bilayer to selectively allow the transfer of solute species 22 while blocking the transfer of solute species 24. Stage 3 of
As a result, solute species 22 transfers from Droplet A to Droplet B while solute species 24 is retained in Droplet A as shown in stage 3 of
In the method of
The transporter species 30 may be a size-selective transporter species—that is, the species 30 may allow transfer of molecules below a certain size and block transfer of larger molecules. Alternatively, the transporter species 30 may be selective in that it permits transfer of solute species having a particular functional group and blocks transfer of solute species that do not include the required functional group. Such a transporter species may be considered as a “passive” transporter species, in that transfer of a species that is permitted to pass by the transporter species 30 is driven by the concentration gradient across the droplet interface bilayer. Alternatively the transporter species 30 may be an “active” transporter species, which can transfer a solute at a greater rate than if driven solely by the concentration gradient and may even transfer solute against a concentration gradient. The transporter species could be a transmembrane protein or other molecule that facilitates the otherwise unfavourable transport of polar compounds or ions through a lipid bilayer, for example as described by Heather E. Findlay, Nicola J. Harris & Paula J. Booth in “In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers”, Scientific Reports 2016, 6, 39349.
The transfer of the solute species 22 from Droplet A to Droplet B may be driven solely by the concentration gradient of the solute species 22 across the droplet interface bilayer, and this may be considered as a ‘passive’ transfer process—as noted, this transfer process will continue until the concentration of the solute species 22 in one droplet is equal to the concentration of the solute species 22 in the other droplet. In other implementations of the method of
In principle, an active transport mechanism—the use of an “active” transporter species and/or the use of an active transfer mechanism—may even allow transport of a target molecule in the direction opposed to the concentration gradient and therefore against the direction of diffusion. Thus, for example, an active transporter species which facilitates an active transport mechanism may be incorporated into the embodiment of
Additionally or alternatively transfer of the solute species 22 may be promoted by providing a sequestering agent for the solute species 22 in the recipient droplet, droplet B. The presence of the sequestering agent is effective to prevent the concentration of the solute species 22 in the recipient droplet increasing, or at least to reduce the rate of increase of its concentration, and thus allow further solute species 22 to be transferred to the recipient droplet before the concentrations of the solute species 22 in the two droplets become equal to one another.
Depending on the intended application of the method of
In the method of
If desired, the droplets may be monitored during the process of transfer of the solute species 22 from Droplet A to Droplet B to determine whether, and if so to what extent, transfer of the solute species 22 is occurring. Examples of techniques for monitoring transport across the droplet interface bilayer include, without limitation:
The method of
The method of
As a further alternative, the process shown in
In the method shown in
In a further modified embodiment, a further solute species, different to solute species 22 and solute species 24, may be introduced into droplet A, by arranging for droplet B to contain the further solute species.
In the method of
While the embodiment of
| Number | Date | Country | Kind |
|---|---|---|---|
| 19182442.4 | Jun 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/067790 | 6/25/2020 | WO |
