WO2016191804A1

WO2016191804A1 - Controlled droplet production and manipulation from a plug using surface acoustic waves

Info

Publication number: WO2016191804A1
Application number: PCT/AU2016/050426
Authority: WO
Inventors: Tuncay ALAN; Adrian NEILD; Muhsincan SESEN
Original assignee: Monash University
Priority date: 2015-06-02
Filing date: 2016-05-31
Publication date: 2016-12-08

Abstract

A microfluidic device for and method of manipulating a plug in a carrier medium, the device including: a substrate; at least one main microfluidic channel for accommodating the plug and the carrier medium, the at least one microfluidic channel having a width and adapted to have fluid flow within; at least one zone extending from the at least one main microfluidic channel; a junction where the at least one main channel and the at least one zone meet; and an acoustic signal source; wherein: the plug and the carrier medium are immiscible; and application or deactivation of an acoustic signal from the acoustic signal source produces a force at an interface between the plug and the carrier medium within the main microfluidic channel, to thereby obstruct the movement of the plug into a particular said zone or to direct at least a portion of the plug through said particular zone or other zone.

Description

CONTROLLED DROPLET PRODUCTION AND MANIPULATION FROM A PLUG USING SURFACE ACOUSTIC WAVES

FIELD OF THE INVENTION

[0001 ] The present invention is generally directed to a microfluidic system, device and method for manipulating a plug contained in a carrier medium using acoustic waves, including surface acoustic waves and standing acoustic waves.

BACKGROUND TO THE INVENTION

[0002] Most recent drug research has been conducted by high throughput screening (HTS). This empirical method exhaustively tests disease-carrying targets (such as cells and proteins) against a library of compounds. Typically HTS reactions are conducted in microtitre plates containing a matrix of wells. Reagents are dispensed into the wells by robotic arms and integrated dispensing pipettes.

[0003] Miniaturization has many advantages in HTS; lowering cost and increasing the number of tests per sample through reduced volumes of reagents. Easier regulation of compound concentration and temperature is achieved due to faster mass and heat transport. However, miniaturised microtitre systems are reaching their limits due to restrictions imposed by the accuracy of robotic dispensing and evaporation in such open systems. This has prompted attempts to design closed digital microfluidics systems using picolitre sized droplets immersed in a carrier medium, because enclosed systems are not prone to evaporation problems and are compatible with sub-microliter volumes.

[0004] However, crucially, the selective combination of multiple reagents, as required by HTS, is still not possible in digital microfluidics due to the continuous droplet production methods used to date. The issue is simple, if there are numerous droplets of one type in a channel and they are merged with numerous droplets of another type produced in a second channel, the reaction of the same two sample types occurs numerous times. Accurate control in droplet production and manipulation is necessary to obtain results of, for example, reactions of different permutations of chemical droplets.

[0005] Digital microfluidics, the compartmentalization of small volumes of one phase within a second immiscible phase, offers the potential of conducting chemical reactions and biological analysis in multiple assays. Hence, offering reduced analysis costs, faster reactions and high throughput analysis capabilities with high sensitivity. However, governing fluid behaviour at the microscale is a challenging task and has led to the investigation of several forcing mechanisms; for example, electric, magnetic, centrifugal and acoustic forces have been used to manipulate flows, droplets, particles and cells at the microscale.

[0006] A desire to control fluid behaviour in digital microfluidic systems has led to the development of special microchannel structures to manipulate droplets in a passive manner relying on hydrodynamic and capillary phenomena. In addition, active manipulation of droplets has been studied. Electric fields are one of the mechanisms that have been utilized to perform such tasks. For example, dielectrophoresis (DEP) has been used to direct droplets into trapping chambers and induce droplet coalescence by destabilizing the oil/water interface. A second actuation mechanism used to gain control over droplet behaviour is acoustic vibration. The vibration can be induced in a number of ways, including the use of a resonating piezoelectric disk controlling the size of bubble produced in a flow- focusing junction or by use of surface acoustic waves (SAW).

[0007] Surface acoustic waves are generated by patterned electrodes on a piezoelectric substrate and are easily integrated to a microfluidic chip. They have been used for particle concentration, trajectory control and sorting for atomization, for sessile droplet displacement and for manipulating cells. In two phase microfluidic systems, SAW has been used for mixing, control of droplet size, individual droplet production, droplet merging and sorting droplets at single and multiple Y-junctions. [0008] The prior art in this field has significant disadvantages. For sorting droplets; Franke et al. [T. Franke, A. R. Abate, D. A. Weitz and A. Wixforth, Lab Chip, 2009, 9, 2625-2627] designed a system such that a single branch had a lower hydrodynamic resistance making that the preferred pathway by default. Ultrasonic forces were utilized to redirect the droplets into the non-preferred, higher resistance path. This has the disadvantage that the channels within the system have to be fabricated in a particular way and the control is not accurate. Furthermore, this method does not offer the ability to divide droplets into smaller volumes.

[0009] Lothar Schmid and Thomas Franke [Saw-controlled drop size for flow focusing Lab Chip 13:1691 -1694, 2013] describe a method for generating different size droplets within microfluidic channels. The droplets are generated from a continuous phase using a standard flow focusing junction. However, when surface acoustic waves are directed into the junction, the droplets 'pinch-off from the continuous phase and different size droplets are produced. This is because the interface is pushed and 'pinch-off of a droplet from a continuous phase takes place before it normally should. This is also described in WO2014066624, where the continuous droplet generation system is able to produce relatively big size droplets. However, it does not offer the ability to create a matrix of observable reactions nor does it allow small droplet sizes to be created from larger ones or allow the ability to create a matrix of observable reactions.

[0010] Another prior art document, US 20150034163 relates to systems and methods for creating droplets, however it does not provide accurate control of droplet size and placement. Nor does it enable a matrix of observable reactions.

[001 1 ] Kaminski et al [Automated generation of libraries of nl droplets. Lab Chip, 12:3995-4002, 2012] discloses generation of nanoliter droplet libraries from larger droplets. Incoming mm-scale droplets are forced through a small orifice to produce nanoliter size droplets. The droplets are spaced and isolated using a third immiscible fluid as a spacer. The spacer is injected into the stream of droplets that are produced after passing through the small orifice to ensure that the droplets are kept separate and don't incidentally join back up together. This operation is not performed on demand, therefore mixing and matching different chemicals is difficult and limited at best. This document describes multiple droplets produced from each plug by injecting a spacer fluid into the stream of plug fluid as it is forced through the orifice. This is undesirable because multiple reactions can occur and the reactions cannot be accurately controlled.

Specifically, individual droplets cannot be produced in this method, only a stream of droplets. Further, individual droplets cannot be produced on-demand or when required by the method described. In addition, this method does not allow droplets from different plugs to be separated and then reacted together when desired.

[0012] Throughout the specification the term plug is used. A plug, as opposed to a droplet, refers to a fluid body which is large enough to be in contact with all four walls of a closed microfluidic channel.

SUMMARY OF THE INVENTION

[0013] It is an object of the present invention to more accurately control droplet production and manipulation and alleviate at least some of the

disadvantages of the prior art.

[0014] According to one aspect of the present invention, there is provided a microfluidic device for manipulating a plug in a carrier medium, the device including: a substrate; at least one main microfluidic channel for accommodating the plug and the carrier medium, the at least one microfluidic channel having a width and adapted to have fluid flow within; at least one zone extending from the at least one main microfluidic channel; a junction where the at least one main channel and the at least one zone meet; and an acoustic signal source; wherein: the plug and the carrier medium are immiscible; and application or deactivation of an acoustic signal from the acoustic signal source produces a force at an interface between the plug and the carrier medium within the main microfluidic channel, to thereby obstruct the movement of the plug into a particular said zone or to direct at least a portion of the plug through said particular or other zone.

[0015] In a preferred embodiment the acoustic signal is a surface acoustic wave signal. It may be a travelling surface acoustic wave signal. Alternatively it may be a standing surface acoustic wave signal.

[0016] The at least one zone may include any one or more of:

(a) one or more reaction chambers;

(b) one or more outflow microfluidic channels;

(c) one or more reaction chambers at an end of one or more outflow microfluidic channels;

(d) one or more reaction chambers extend from one or more of the outflow microfluidic channels;

(e) one or more outflow microfluidic channels are reaction chambers extending from the main microfluidic channel.

[0017] In the device, the junction may be a reaction chamber.

[0018] In the device, an acoustic mismatch may occur at the interface of the plug and the carrier medium; and wherein the movement of the plug occurs because the interface of the plug and the carrier medium is displaced.

[0019] The substrate of the device may be a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.

[0020] The device may have one main microfluidic channel and two zones which are outflow channels forming a Y-junction.

[0021 ] The carrier medium may be a fluid and preferably water. The plug may be a form of oil and is preferably also a fluid. [0022] Preferably when the acoustic signal is applied to the device it causes a portion of the plug to split. Alternatively, if the signal is applied before the plug enters the main channel, when the acoustic signal is no longer applied to the device, termination of the signal preferably causes a portion of the plug to split.

[0023] According to another aspect of the present invention, there is provided a method of manipulating a plug in a carrier medium through microfluidic channels using a device having: a substrate; at least one main microfluidic channel for accommodating the plug and the carrier medium; at least one zone extending from the at least one main microfluidic channel, the at least one microfluidic channel having a width and adapted to have fluid flow within; a junction where the at least one main microfluidic channel and the at least one zone meet; and an acoustic signal source, the method including:

introducing into the main microfluidic channel a solution including the plug and the carrier medium, wherein the plug and the carrier medium are immiscible fluids; and

applying or deactivating an acoustic signal source to produce a force at an interface between the plug and the carrier medium within the main microfluidic channel, to thereby obstruct the movement of the plug into a particular said zone or to direct at least a portion of the plug into said particular zone or other zone.

[0024] Preferably the plug is controllably split into at least one droplet by applying the acoustic signal to the plug at the junction, directing the droplet into the particular zone.

[0025] Alternatively, the method may include a further step of applying an acoustic signal prior to the plug and the carrier medium being introduced into the main channel and wherein the plug is controllably split into at least one droplet by deactivating the acoustic signal when the plug is at the junction, directing the droplet into the particular zone. [0026] Preferably the acoustic signal is a surface acoustic wave signal. The acoustic signal may be a travelling surface acoustic wave signal. Alternatively, the acoustic signal may be a standing surface acoustic wave signal.

[0027] The at least one zone may include any one or more of:

(a) one or more reaction chambers;

(b) one or more outflow microfluidic channels;

[0028] The plug may be controllably split into multiple daughter droplets by adjusting the input power of the signal source.

[0029] At least one daughter droplet may be directed into a zone and at least a second daughter droplet may be directed into a second zone which is different from the first zone. The daughter droplets may be of uneven volume.

[0030] Preferably the carrier medium is a fluid.

[0031 ] The substrate may be a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.

BRIEF DESCRIPTION OF THE DRAWINGS [0032] The present invention will now be described with reference to the following drawings which illustrate experiments conducted using the present invention. It is to be appreciated that the present invention is not limited to the experimental examples and that other embodiments are also envisaged.

Consequently the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.

[0033] In the drawings:

[0034] Figure 1 (a) shows a microfluidic device for conducting a prior art method. Figure 1 (b) shows an enlarged version of a section of the microfluidic device of Figure 1 (a).

[0035] Figure 2 shows a prior art system and device for conducting a prior art method.

[0036] Figures 3(a) and (b) show what occurs when the method of the present invention is not applied.

[0037] Figure 4(a) shows the plug manipulated by the method of the present invention. Figures 4(b)-4(e) show time lapse images of Figure 4(a).

[0038] Figure 5 shows multiple exposure images of the plug having a prior art method applied.

[0039] Figures 6(a)-(f) show time lapse images of the plug having a prior art method applied.

[0040] Figures 7.1 (a)-(f), 7.2(a)-(f) and 7.3(a)-(f) show time lapse images of a plug manipulated by a prior art method at different power intensities. [0041 ] Figure 8 shows a plot of the volume ratio of two daughter droplets divided out of a plug at a give power level according to the method of the present invention.

[0042] Figure 9 shows plug velocity verses power when the plug is subjected to the method of the present invention.

[0043] Figure 10 shows plug velocity verses power when the plug is subjected to the method of the present invention.

[0044] Figure 1 1 shows an embodiment of the present invention.

[0045] Figure 12 shows an embodiment of the present invention.

[0046] Figure 13 shows an embodiment of the present invention.

[0047] Figure 14(a) shows a prior art method and Figure 14 (b) shows an embodiment of the method of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENT

[0048] The following description in conjunction with the accompanying drawings describes various examples of the method, device and system used to control plug manipulation and splitting using acoustic waves according to embodiments of the present invention.

[0049] Digital microfluidic systems, in which isolated droplets are dispersed in a carrier medium, offer a method to study biological assays and chemical reactions highly efficiently. However, it is challenging to manipulate these droplets in closed microchannel devices. In the present invention, a method and system is described to selectively manipulate plugs (droplets with diameters larger than the channel's width) at a specially designed Y-junction within a microfluidic chip. The method and system make use of acoustic waves, and in one embodiment, surface acoustic waves (SAWs) impinging on a multiphase interface in which an acoustic contrast is present. As a result, the liquid-liquid interface is subjected to acoustic radiation forces. These forces are exploited to steer plugs into selected branches of the Y-junction. Furthermore, the input power can be finely tuned to split a plug into two uneven plugs or droplets. The plugs can be manipulated as a whole or in other manners, based on plug volume and velocity. The experimental results indicate that there is a threshold plug volume after which the steering requires elevated electrical energy input. This plug manipulation method is easily integrated into existing lab-on-a-chip devices and it offers a robust and active plug manipulation technique in closed microchannels.

[0050] The term plug, as opposed to a droplet, refers to a fluid body which is large enough to be in contact with all four walls of a closed microfluidic channel. In the case of a droplet in a channel the nature of the acoustic forces generated on it are similar to those for a solid particle. The acoustic radiation force is determined by integrating second order time averaged terms over the surface of the (solid or fluid) sphere. In the case of a plug, however, the ultrasonic interaction is restricted to the interface between the two immiscible fluids, in a straight channel this is the head and tail of the plug, around a junction an additional surface is created at the opening of the second channel. As such, in order to steer a plug, the liquid/liquid interface, at which there is an acoustic impedance mismatch, must be displaced.

[0051 ] Generally, this invention relates to manipulating plugs in junctions of equal hydrodynamic resistance using acoustically generated forces. The junctions discussed are Y-junctions, that is, one inlet channel and two outlet channels, however, any number of input and output channels can be used and hence the junction can be of different shapes and configurations. [0052] This invention further generally relates to directing focused surface acoustic waves (SAWs) at the plug/carrier medium interface (for example, oil/water interface), inducing a net acoustic radiation force sufficient to obstruct the progress of a plug into a selected branch of the Y-junction. Not only does this method allow an incoming plug to be steered into the desired branch at the Y- junction, the incoming plug is able to be controllably split into two daughter plugs or droplets of uneven volume by adjusting the input power of the SAWs.

[0053] The method of the present invention is used to affect and manipulate the plug in a carrier medium. The method and system of the present invention can be used to easily carry out and observe reactions of different samples in an array or matrix type arrangement.

[0054] The system of the present invention includes a microfluidic device for manipulating a plug in a carrier medium. The device includes a substrate on which there is at least one main channel. There may be multiple main channels included in the device. One or more zones extend from the at least one main channel. The zones may include secondary microfluidic channels, reaction chambers where reactions of different chemicals take place or any other type of area to observe or undertake reactions. The device also has an acoustic signal source for producing an acoustic signal, such as a travelling surface acoustic wave or a standing surface acoustic wave.

[0055] As shown in Figure 1 1 , in the device 1 , one or more reaction chambers 4 are located off and extend from the main microfluidic channel 2. As a plug 5 of the required chemical passes an outflow channel 9 to a reaction chamber 4 a small portion or droplet 15 of the plug 5 is split off when SAWs 3 are applied to the plug 5 / carrier medium 10 (fluid) interface.

[0056] In this method, firstly, samples for reaction or observation in the form of plugs in a carrier medium fluid are introduced into a main channel of a microfluidic system. Introducing the samples (as a plug) can be achieved through the use of existing technologies. The plugs may be chemicals, fluids or other reactants. The plugs introduced into any one system may be all the same sample. Alternatively, the plugs may all be different samples. Further, the plugs introduced into the system may be any combination of samples.

[0057] The method then allows for a portion of the plug to be split from the plug. If the portion split from the plug is small (for example, picoliter size) and is to be further manipulated, for example in a reaction, it is termed a reaction droplet. Alternatively, if the portion split from the plug is large, typically micro or nanolitres in size, it is termed a daughter plug. A daughter plug is a plug that has been split from the original plug and can be further split in the system to form reaction droplets which are used in reactions in the system.

[0058] Splitting the reaction droplet from the plug is achieved by using acoustic waves. The acoustic waves control how the plugs are split. The plugs are split on-demand, that is, when the user wants a portion of the plug split off. This could be either as a reaction droplet or a daughter plug depending on the application or requirement.

[0059] A reaction droplet is split from a plug and directed into another channel or into a reaction chamber. Alternatively, the reaction droplet may be directed into another area or zone of the system.

[0060] As a plug containing a particular fluid, chemical or sample passes a zone into which the chemical is required, a reaction droplet is split off the plug and directed into that zone. This is achieved in one embodiment by an acoustic wave signal which is activated and applied as the plug passes a particular zone. The acoustic signal directs the reaction droplet into the zone. Once in the zone the droplet can react with other droplets, chemicals or reactants already in the zone. Alternatively the droplet may need to wait until other droplets, chemicals or reactants are introduced into the zone. The zone may be another microfluidic channel, a reaction chamber or any other area for which the droplet of a chemical or reactant is required.

[0061 ] The acoustic wave applied to the plug may be a surface acoustic wave. In a preferred embodiment, surface acoustic waves (SAW) affect the behaviour of the boundary of the plug. This can be in the form of deforming the boundary of the plug in the carrier medium (that is, the plug / carrier medium interface) so that a reaction droplet is split from the original plug. Alternatively, in another embodiment, a propagating SAW can be used to push at the plug / carrier medium interface such that a droplet is removed from the plug as it moves through the system. In an embodiment which uses a standing acoustic wave, the "tail" of the plug is held back until the plug stretches and divides into a plug and a small reaction droplet.

[0062] In another embodiment, acoustic waves are activated and applied to the device prior to the plugs and carrier medium being introduced into the system. As the plugs carrying a required chemical pass a zone into which it is required, deactivating or turning off the acoustic wave signal temporarily causes a reaction droplet to split from the plug and is directed to the required zone. As such, by activating or deactivating the acoustic wave signal, the user has full control over which portions of which plugs will be directed into which reaction chamber - multiple permutations of droplets and reactions can be easily tested.

[0063] As shown in Figure 1 1 , different plugs 5, 8 pass the reaction chambers 4, 44. If a chemical in a plug is required in a particular reaction chamber, application of SAWs will cause a droplet to split from the plug into the particular reaction chamber. Figure 1 1 shows reaction chamber 4 into which droplet 15 has been split from plug 5 as it moves through the channel 2. Droplet 18 has been split from plug 8 as it moves through the channel 2. Reaction chamber 44 shows droplets 18 and 19, split from plug 8 and 9 (not shown), respectively, by application of SAWs 33. [0064] Figure 1 1 shows splitting of droplets from a moving plug to reaction chambers on demand using travelling focussed SAWs. If a droplet from a plug needs to be deposited into a particular reaction chamber, the SAW is activated when the correct plug passes the opening of the reaction chamber, causing a droplet to split from the plug.

[0065] Figure 12 shows splitting of a droplet 122 from a moving plug 121 when there is a restriction in the main microfluidic channel 2.

[0066] In Figures 1 1 , 12 and 14b SAWs affect the behaviour of the boundary of the plug, deforming the boundary of the plug so that a droplet is split out from the plug. The propagating SAW pushes the interface such that a droplet is removed from the moving plug.

[0067] Figure 13 shows an alternative method. In this method a droplet 1 13 is split from a moving plug 123 at its tail. This is achieved by using standing surface acoustic waves. In this method the tail of the plug is held back until the plug stretches and snaps into a plug 123 and a small droplet 1 13.

[0068] Figure 14a shows a plug approaching a junction will not split under certain conditions. As the plug approaches a junction it will attempt to enter a channel that branches from the main channel. However, as the plug moves along the main channel, that part of the plug that attempted to go into the branch will be forced out of the branch and into the main channel. No droplet splitting has occurred.

[0069] However, Figure 14b shows what happens if surface acoustic waves are applied to the same moving plug. In this figure it is clear that as the plug passes the junction a SAW is applied and a droplet is split from the moving plug and directed into the channel that branches from the main channel.

[0070] Experimental Methods [0071 ] A microfluidic chip and system designed for manipulating and steering plugs is shown in Fig. 1 (a). Figure 1 (a) shows polydimethylsiloxane (PDMS) microfluidic channels bonded onto a piezoelectric substrate with pattered electrodes, the two inlets (an oil and a water phase) are connected by a T- junction (not visible) at which plugs are formed upstream of the Y-junction. Figure 1 (b) is an enlarged view of the Y-junction of Figure 1 (a). In this Figure, the Y- junction is seen with connecting channels between the two downstream

branches, the two sets of electrodes focussed at the neck of the junction are also shown.

[0072] Water-in-oil plugs are generated via the use of a T-junction geometry just after the inlets (an oil and a water phase), upstream from the Y junction. After formation, plugs travel downstream along a 100 μιη wide and 50 μιη high rectangular cross-section microchannel, until they reach the Y-junction which is positioned at the centre of the chip for easy optical access (Fig. 1 b). The three channels that meet to form the Y-junction are of the same dimensions. Downstream of the junction, connecting channels pass between the two emerging branches. In contrast to changing the direction of a droplet at a Y-junction, steering a plug will alter the fluid flow profiles throughout each outlet channel. These connecting channels are designed to assist with the equalization of pressure in the two branches. Focused electrode pairs deposited onto a piezoelectric substrate (inter-digital transducers) were aligned so that the focal area coincides with the neck of the Y-junction.

[0073] Fluorinated oil (FC-40) stabilized with 2% (w/w) surfactant (Pico- Surf™1 , Dolomite) (viscosity, μ = 3:4 cP, interfacial tension γ ~ 5 imN/m) was used as the continuous phase while the dispersed phase was deionised (Dl) water. Syringe pumps (NE-1000, New Era Pump Systems, Inc.) were used to regulate fluid flow as desired. The syringes were connected to the device using PTFE tubing. A 3D-printed platform was used to clamp the device as well as to interface with the electrodes on the piezoelectric substrate. A microscope (Olympus BX43, Tokyo, Japan) equipped with an eyepiece camera (5MP, Dino-Lite AM7023B, New Taipei City, Taiwan) was used for image acquisition. The videos were analysed using "Droplet morphometry and velocimetry"(DMV) software developed by Basu. DMV is a video processing software that makes use of edge detection and droplet tracking to extract information about the droplets such as shape, velocity, size, etc. In the experiments described herein, DMV analysis was carried out using the same settings over a range of videos to obtain plug velocity and size information.

[0074] A power signal generator (F20, PowerSAW) (BelektroniG, Bruenig & Guhr Elektronik) was used to generate SAWs on the piezoelectric substrate by applying an AC signal to the inter-digital transducers. The PowerSAW determines the scattering parameters during operation which are used to accurately calculate the actual power (accounting for any losses in the cabling) that a device is using to induce the SAWs.

[0075] Focused inter-digital transducers (FIDTs) with a pitch of 60 μιη operated at 64 MHz in the experiment described. FIDTs consist of curved electrodes and they have been used widely in prior art to focus the ultrasonic power along a narrow region, which in the presently described system coincides with the neck of the Y-junction.

[0076] In order to fabricate the FIDTs, lithium niobate, LiNbO₃ (LN), substrates of 500 μιη thickness (single side polished, 128°Y-cut, X-propagating) were patterned and a 200nm thick aluminium layer was deposited following a 10nm chromium layer for adhesion. The substrate was then diced into the desired dimensions. Similar to the microchannels, polydimethylsiloxane (PDMS)

(SYLGARD ® 184, Dow Corning) (10:1 ) was cast onto a silicon master mould patterned by standard lithography followed by deep reactive ion etching to a depth of 50 μιη and silanization. Both the LN substrate and PDMS surfaces were subjected to air plasma (Harrick Plasma PDC-32G) for surface activation and subsequent covalent bonding using a custom built alignment system operated under a stereo microscope (Olympus, SZX16, Tokyo, Japan). [[00007777]] OOppeerraattiinngg PPrriinncciippllee

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[0080] where E, is the energy density of the incident wave, R_c is the acoustic reflection coefficient and c is the speed of sound in the fluids.

[0081 ] By use of forces generated at the interface between two immiscible liquids, a plug is directed to exit a Y-junction along a selected branch. As a consequence of the motion of the plug through the junction, the flows in the attached channels are affected. To understand this process, the system whilst a plug is being steered, as shown in Figure 2, is analysed.

[0082] Microfluidic networks can be considered analogous to a resistor network in an electrical circuit to simplify analysis. Considering capillary and Reynolds numbers are small, a plug that is being steered, as shown in Figure 2, can be analysed using the following set of equations: [0083] Pi - RiQi + 2γΗί = _Vi (Equation 2)

[0084] Pu + RuQu + 2YH_U + PARP u (Equation 3)

[0085] P_l + R_lQ_l + 2_YH_l = _Pl (Equation 4)

[0086] where P and p are local and external pressures respectively with subscripts for inlet (i), upper (u) and lower (I) channel (the descriptions being with reference to Figure 2); Q denotes flow rates, γ is the interfacial tension between the two mediums and /-/ represents the mean curvature of the interface. P_ARP is the pressure originating from acoustic radiation acting on the interface (referred to as acoustic radiation pressure), it is applied in the equation, by the upper electrodes to steer the plug into the lower channel, and R stands for the microfluidic analogy of ohmic resistance, or hydrodynamic resistance. Keeping in mind that the capillary pressure will be the same for the steering channels because their dimensions are equal (that is, yH_u = γΗ ). Subtracting equation 4 from equation 3 to get:

[0087] P_ARP = Ap— AP— AM (Equation 5)

[0088] where Ap and ΔΡ are local and external pressure differences (that is, Ap = Pu -Pi) and AM \s the difference of pressure drops in the upper and lower channels (that is, AM = R_UQ_U _ RiQi). The local pressure difference between the two interfaces (Ap) can further be simplified considering a single-phase

microfluidic system, in which case, the pressure drop in a microfluidic channel of rectangular section is known. Substituting this into equation 5 to get:

[0089] P_ARP = A\J_mLp— AP— AM (Equation 6)

[0090] where A is a constant depending on the channel geometry, U_m is the mean flow velocity, L is length and μ is the viscosity of the fluid. This is the governing equation which indicates how much acoustic radiation pressure will be needed to steer a plug under a set of given circumstances which we will be analysed.

[0091 ] Fluid pumped through the inlets

[0092] Depending on the way flow is controlled through the junction, further simplifications can be made. Firstly, if the flow is pumped through the inlets, then both outlets are at an equal pressure (open to atmosphere) (that is, ΔΡ = 0). Furthermore, in such a system, once the head of a large plug has passed the entrance to all the connecting channels, there will not be any flow in the upper channel, hence Q_u = 0 and consequently Qi = Q,. Applying these factors in the governing equation 6, obtains:

[0093] P_ARP = Αυ^μ + (Equation 7)

[0094] This suggests that the acoustic radiation pressure required to impede the progress of a plug is equal to the pressure drop from the top channel interface to the bottom channel interface of the plug (first term in equation 7) and an additional hydrodynamic resistance term (second term in equation 7). If an order of magnitude analysis is performed on the terms that contribute to the acoustic radiation pressure requirement, the hydrodynamic resistance term is an order of magnitude higher than the pressure drop term when fluid is being pumped through the system. This implies that the necessary acoustic power depends highly on the hydrodynamic resistance in the steered channel as well as the inlet flow rate.

[0095] Fluid withdrawn from outlet channels

[0096] An embodiment when the connecting channels are not blocked is described. If the flow is drawn through the system, as opposed to fluid being pumped through the inlets, the previous assumptions do not hold. It is this flow regime that was investigated with embodiments of the system of the present invention predominantly, as in a more complex digital microfluidic system, more complex flow regimes are expected rather than results from simply pumping a constant volumetric flow rate through a single junction.

[0097] To draw the fluid, the two syringes attached to the outlets will attempt to maintain equal flow rates in the upper and lower channel, hence

2Q_U = 2Q| = Qj. Even when a plug is being manipulated or steered, before the connecting channels are blocked, flow will be equal in both outlet channels as dictated by the syringe pumps. This is because the continuous medium is able to pass into the upper branch from the lower branch through the connecting channels. In such a case, the working outlet pressures of the syringes are reasonably close (that is, AP ~ 0). Moreover, it is further assumed that the hydrodynamic resistances in the outlets have a static and a dynamic term, for continuous medium and the plugs in the outlet, respectively:

[0098] R = R + nR_p (Equation 8)

[0099] Where R is the average hydrodynamic resistance of a channel without any plugs and n is the number of plugs currently in the channel and R_p is the hydrodynamic resistance of a plug, assuming the plugs are the same. Applying all the above assumptions and substituting equation 8 to the governing equation 6 for the system when the connecting channels are not blocked by the plug and the syringes are equally withdrawing from the outlets, obtains:

[00100] P_ARP = Αυ^μ + (nR_p)Qi (Equation 9)

[00101 ] For every plug that is steered into the desired channel, there will be an additional R_pQj pressure requirement where R_p is the hydrodynamic resistance of that plug. An order of magnitude study on these governing terms reveal that these terms are of the same order, implying that they are equally important factors for steering plugs through a Y-junction. [00102] An embodiment when the connecting channels are blocked by the plug is now described. Once the head of the plug reaches the end of the connecting channels, then fluid flow ceases to exist in the upper channel until the tail of the plug reaches the neck of the junction. In this stage (only a very short time), the pressure in the syringe connected to the upper channel decreases rapidly as the constant suction of the syringe expands any air in the syringe or results in compliance within the tubes or PDMS. A rapid rise in the required PARP is expected as the length of the plug increases beyond the length of channel over which connecting channels are present.

[00103] Results and Discussion

[00104] It is well known that a long plug will split into two equal sized plugs upon entering a two way junction with equal hydrodynamic resistances. Figure 3a shows a plug that is split into two at the Y-junction without any acoustic energy applied. This is explained by the equal amount of pressure encountered by the interfaces of the plug (that is, p_u ~ /¾). The multiple exposure image shows the symmetric advancement of the interface into the outlet channels (Figure 3b).

[00105] In contrast, by exciting surface acoustic waves, the progression of an interface into either of the branches can be halted and stabilized at the junction as a result of the net acoustic radiation pressure (ARP) induced on the interface itself. This is demonstrated in Figure 4a for the case in which fluid is pumped through the channel with both outlets at atmospheric pressure. As explained above in the section titled 'fluid pumped through the inlets', this flow scenario enables variation in the flow rates in each exit channel enabling an expectation that longer plugs can be steered. Indeed it can be seen that actuating the top electrode blocks the upper channel (as shown in Figure 4a) for the full duration of the time required for the plug to completely pass through the junction. Of course, meanwhile the leading interface progresses along the lower channel (see Figure 4c). As a result, a plug with a relatively large volume, here 7.33nl_, can be steered intact, into the desired outlet. [00106] Steering of these large plugs is only possible when both the outlets are open to atmosphere and the fluids are pumped from the inlet ports. For practical purposes, the analysis carried out in the rest of this work focuses on a different case where fluid is withdrawn at equal flow rates from each outlet using syringes mounted on a double syringe pump. This method is likely to be more applicable to situations in which additional manipulations such as merging or additional steering are required further downstream of the Y-junction so a pressure balance across the branches can not be assumed.

[00107] The suction induces fluid flow in both outlet channels at all times and therefore restricts the maximum volume of the plug that can be steered. When both outlets are open to the atmosphere, a scenario involving absence of flow in one of the channels is possible. However when suction is present, this is not the case. The connecting channels between the two branches are designed to allow the carrier medium to flow from the branch into which the plug is steered to the other branch allowing an equal flow condition in both branches to be maintained until the connecting channels are blocked.

[00108] The effect of the connecting channels can be observed by analysing a series of multiple exposure images. Initially, under the influence of the acoustic waves, the interface is observed to be held in a stable location (see Figure 5a) up until the head of the plug blocks the last available connecting channel. At this point, the stability of the interface is quickly lost. The syringe pump connected to the upper channel seeks to extract fluid continuously and as a result the interface is drawn, against the resistance of the acoustic forces, into the lower branch (see Figure 5b).

[00109] The time-lapse images of a plug during successful manipulation and steering are shown in Figure 6. As the plug reaches the Y-junction, it

preferentially follows the upper channel due to the additional acoustic pressure applied to the interface via the impinging SAWs from below (see Figure 6b). It can be observed that the plug does not bulge into the bottom channel until it totally blocks the connecting channels (see Figure 6c). When the connecting channels are free, oil passes from the upper branch to the lower one such that a constant flow is maintained in both outlets and allowing, with the assistance of the acoustic radiation pressure, a stable interface at the entrance to the lower channel. However, when the plug blocks the connecting channels, the pressure starts to decrease in the lower channel (as a result of the absence of flow) and the interface at the junction of this lower channel starts to advance (see Figure 6d). The plug progresses in the lower channel and develops a finger until an opening 'tunnel' forms as the tail end of the plug reaches the junction (see Figure 6e) allowing oil flow into the lower channel. Below a certain finger length, interfacial tension can draw the plug back into the upper channel and to its minimum energy state (see Figure 6f) leading to successful steering. If the finger extends further into the channel, the plug ultimately splits, this limits the maximum volume of a plug that could be steered under these conditions.

[001 10] In the system presented here, if the plug was longer than the one shown, the tail of the plug would be further upstream in Figure 6d, delaying the possibility of a tunnel forming, and as such the interface would progress further into the lower channel eventually causing the plug to split.

[001 1 1 ] Analysing the plug manipulation and steering at various acoustic powers the development of the finger (formed by the movement of the interface into the undesired channel) is postponed as we switch from low to high acoustic energy (see Figure 7). Higher acoustic energy induces a higher ARP on the interface and thus compensates for the decreasing pressure at the upper channel. Whereas lower ARP is unable to restrain the interface sufficiently and the plug develops a longer finger into the upper channel which eventually leads to the break-up of the plug (in both the low and medium power cases shown).

[001 12] This is advantageously used in manipulating the applied electrical input power to control how much the plugs split. In Figure 8, the volume ratio

{MAX{ Vi, V₂)/{ V1+ V2)), where V ^'\s the volume of the daughter plugs) is shown with respect to the applied input power. It can be seen that for plugs of 2.35nl_ mean volume (relative standard deviation (RSD) = %20.2) and θδθμιη/ε mean velocity (RSD = %21 .6), the splitting ratio increases as the applied power increases. This is because of the elevated net acoustic radiation pressure applied on the interface. It is able to hold the interface for a longer period of time allowing the size of the manipulated and steered daughter plugs to increase up to a point where the plug is completely steered into the desired branch. With this, an incoming plug could be split in half, split into two uneven plugs controllably or steered into one of the channels as a whole, on demand. This method of controlling the manipulation of a plug or droplet is more commercially valuable than merely the method of steering the plug.

[001 13] The control which can be gained by SAW on the behaviour of the plug interfaces at the junction has been examined as well as the various resultant outcomes in terms of steering or plug break-up. The key parameters governing this process will now be examined, namely the plug volume, velocity and the applied electrical power. Data has been gathered over a range of plug sizes and velocities by altering the flow conditions imposed by the three syringe pumps (one on the oil inlet, and one on each of the two outlets). The reported plug velocity and volume values are measured using the DMV video analysis software, whereas the power input and S1 1 values were extracted from the signal generator, PowerSAW.

[001 14] To characterize how these parameters influence the steering process, the maximum plug volume that can be steered by different electrical power inputs is plotted (see Figure 9a) for a subset of data exhibiting relatively low plug velocity variance (mean velocity of 1007μιη/5 and relative standard deviation (RSD) of %17.7), hence restricting the effect of this variable to a minimum. Applying a linear fit to this data, an approximating function for correlating plug volume and required power input with an R-squared value of 96.8 is obtained. This fitted line is later used to remove the effect of plug volume from the data set used in Figure 9b. [001 15] For each data point in Figure 9b, the velocity is plotted against the power; the power being first normalized based on the plug volume using the linear fit function obtained from Figure 9a. Across the data set, two outcomes can occur, steering or splitting of the plugs; the former are displayed as green square markers, whereas the latter are depicted using red circular markers. The error bars shown are standard deviations of the measured velocities calculated by the DMV software.

[001 16] Three regions emerge from the scatter plot; one in which steering successfully takes place, one where the plugs are manipulated and split and the intersection of these regions is the transition region where steering is not always successful. This is attributed to the slight hydrodynamic resistance variance in the outlet channels. Clearly as the velocity of a plug approaching the junction increases, the acoustic energy required to stabilize its interface and steer it also increases. From the previous analysis, if equal hydrodynamic resistance in both channels is assumed, the required acoustic radiation pressure increases proportional to the mean flow velocity (that is, PARP ⁰⁰ U_m) (Equation 9). The input electrical energy (P_e), on the other hand, is proportional to the acoustic radiation pressure induced in the working fluids, squared (that is, Pe ^QC PARP²) which leads to a square relationship between electrical power input and the plug velocity. For this reason, the boundaries of the transition region between steering and splitting (Figure 9b) have been marked by using two approximating functions of the form P_e = AiUm² where A is a constant value selected to identify the upper and lower bounds of the transition region.

[001 17] The final set of data in Figure 10, shows the required power as a function of plug volume. Again to remove the effect of the third variable, in this case the velocity of the plug, the power has been normalized, here by using the fitted curves in Figure 9 (it can be seen in Figure 10 that the data tends to sit in different power bands, each band is a different power tested, the height of each band gives an idea of the modest change this normalization step makes).

Specifically, the slope of these curves was used to perform a linear interpolation for the power input values relative to a band of plug velocity values

RSD=%26.8). The results are plotted in Figure 10 where, again, the green square markers identify plugs that were steered as a whole and the red circular markers are for plugs that split. The insets provide images for various plug volumes in the dataset so that the length of the plugs can be visually compared with the length of the channel over which the connecting channels are present.

[001 18] At low volumes all the plugs steer even at low power inputs, however once the volume is increased such that the plug is long enough (^« 1 .5nl_) to block all the connecting channels, an intact plug can no longer be steered without substantially increasing the power. This clearly demonstrates the role the connecting channels play, and ties in with the description of the pressure characteristics in the channels given above. For larger plug volumes, the interface at the entrance to the undesired channel becomes harder to stabilize due to the suction of the syringe pump attached to that channel, thus necessitating a significant increase in power.

[001 19] A transition region where some plugs are successfully steered and some are split is also shown in Figure 10. This is attributed to the hydrodynamic inequalities in the outlet channels. The largest plug (^« 2.62nl_) that was

successfully steered (see inset of Figure 10) was limited by the design of the connecting channels. However, having established the functionality of the connecting channels, it is clear that it is possible to steer plugs of larger volume by increasing the number of connecting channels after the Y-junction. The smallest plug that was steered, on the other hand, was simply limited by the droplet generating T-junction geometry which could easily be modified to produce smaller plugs or droplets.

[00120] Conclusion [00121 ] A novel microfluidic lab on a chip device that is able to steer plugs of 0.75nl_ to 2.62nl_ volume into the desired branch of a specially designed Y- junction is presented. Focused surface acoustic waves act on the interface of water-in-oil plugs which results in a net acoustic radiation pressure applied at the interface. This pressure deforms the interface and forces plugs to steer into the selected branches of the Y-junction. The system is characterized and establishes operating regions for the successful steering of plugs with various volume and velocities. In addition, the manipulation and splitting of the plugs can be

modulated to produce daughter plugs of desired volume. This offers additional versatility to the described plug manipulation and steering method. This is useful in droplet microfluidic systems where active sorting of plugs is required. Surface acoustic wave generating IDTs are easily integrated onto existing lab on a chip devices and can be coupled with other droplet microfluidic manipulation methods like merging, mixing and dilution.

[00122] Advantages of the present invention over the prior art

[00123] The prior art methods and systems relating to creating droplets in digital microfluidic systems, all have disadvantages and limitations making them unsuitable for digital microfluidics. None of the currently available methods allow for controllable droplet production from a moving plug, and hence a matrix of multiple reaction chambers would not be possible. The present invention enables increased accuracy in droplet production and manipulation, which is necessary to obtain different permutations of outcomes.

[00124] Additional advantages of the present invention include the use of surface acoustic waves (SAW) to affect the behaviour of the boundary of the plug. This can be carried out by deforming the boundary so that a daughter droplet is produced by using the propagating SAW to push the interface such that a droplet is removed from the moving plug. Or alternatively a standing acoustic wave is used to hold back the tail of the plug until it stretches and a portion of the plug is split off into a small daughter droplet. [00125] A further advantage of the present system and method is the on- demand droplet manipulation of large droplet sizes. Current technologies enable continuous droplet generation for relatively big size droplets, but do not enable the ability to create a matrix of observable reactions. Other available

technologies provide for smaller droplets but do not combine this with an on- demand operation, hence mixing and matching different chemicals would be limited.

[00126] Furthermore, the presently described method and system allows a controlled break up of a plug. This means that the main body of the sample (the plug) can be pumped around the channel and broken up at the location of the reaction chamber, negating any issues with bringing the drop to the right location. The present invention advantageously provides a system which can break up a moving plug, ejecting a small daughter droplet at, and only at, desired locations. These locations would be at the entrance to reaction chambers, a term used only to indicate that the reaction would take place adjacent to the main channel. The present invention is advantageous and extremely desirable to allow HTS to be incorporated onto digital microfluidics on a microfluidic chip.. This is unlike in WO2014066624 where it allows controlled "pinch off" of a droplet from a continuous phase.

[00127] Further advantages of the present invention include the use of surface acoustic waves to affect the behaviour of the boundary of the plug; the use of SAW to control plug splitting and droplet formation; the use of standing acoustic waves to stretch the plug and form a daughter droplet; the ability to form a daughter droplet from a moving plug rather than a static one. The ability to create a matrix of observable reactions, and hence simultaneous multiple permutations; greater accuracy and control of droplet size; and greater accuracy and control of droplet placement. A typical application for this method would be in high throughput screening for drug research. [00128] The present invention produces a single droplet from a plug, rather than multiple droplets, but more importantly the method and system of the present invention allows the production of a droplet from a plug to take place when desired by a user rather than automatically. If a series of plugs are circulating around a chip, the present invention allows removal of droplets from certain plugs and reacting them together in various reaction chambers. Each reaction chamber has a transducer capable of generating a SAW such that the break off can take place when desired. The prior art only allows a constant stream of droplets to be formed from the plug. The prior art does not allow this On-demand' production and manipulation of droplets and plugs.

[00129] Variations can be made to the above-described arrangements without departing from the spirit or scope of the invention as described herein or as claimed in the appended claims.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1 . A microfluidic device for manipulating a plug in a carrier medium, the device including:

a substrate;

at least one main microfluidic channel for accommodating the plug and the carrier medium, the at least one microfluidic channel having a width and adapted to have fluid flow within;

at least one zone extending from the at least one main microfluidic channel;

a junction where the at least one main channel and the at least one zone meet; and

an acoustic signal source;

wherein:

the plug and the carrier medium are immiscible; and

application or deactivation of an acoustic signal from the acoustic signal source produces a force at an interface between the plug and the carrier medium within the main microfluidic channel, to thereby obstruct the movement of the plug into a particular said zone or to direct at least a portion of the plug through said particular zone or other zone.

2. A microfluidic device according to claim 1 , wherein the acoustic signal is a surface acoustic wave signal.

3. A microfluidic device according to claim 2, wherein the acoustic signal is a travelling surface acoustic wave signal.

4. A microfluidic device according to claim 2, wherein the acoustic signal is a standing surface acoustic wave signal.

5. A microfluidic device according to claim 1 , wherein the at least one zone includes any one or more of:

(a) one or more reaction chambers;

(b) one or more outflow microfluidic channels;

6. A microfluidic device according to claim 1 , wherein the junction is a reaction chamber.

7. A microfluidic device according to claim 1 , wherein an acoustic mismatch occurs at the interface of the plug and the carrier medium; and wherein the movement of the plug occurs because the interface of the plug and the carrier medium is displaced.

8. A microfluidic device according to claim 1 , wherein the substrate is a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.

9. A microfluidic device according to claim 1 , wherein there is one main microfluidic channel and two zones which are outflow channels forming a Y- junction.

10. A microfluidic device according to claim 1 , wherein the plug is a form of oil and the carrier medium is water.

1 1 . A microfluidic device according to claim 1 , wherein when the acoustic signal is applied to the device it causes a portion of the plug to split.

12. A microfluidic device according to claim 1 , wherein when the acoustic signal is no longer applied to the device, termination of the signal causes a portion of the plug to split.

13. A method of manipulating a plug in a carrier medium through microfluidic channels using a device having: a substrate; at least one main microfluidic channel for accommodating the plug and the carrier medium; at least one zone extending from the at least one main microfluidic channel, the at least one microfluidic channel having a width and adapted to have fluid flow within; a junction where the at least one main microfluidic channel and the at least one zone meet; and an acoustic signal source,

the method including:

14. A method according to claim 13 wherein, the plug is controllably split into at least one droplet by applying the acoustic signal to the plug at the junction, directing the droplet into the particular zone.

15. A method according to claim 13 further including the step of applying an acoustic signal prior to the plug and the carrier medium being introduced into the device and wherein the plug is controllably split into at least one droplet by deactivating the acoustic signal when the plug is at the junction, directing the droplet into the particular zone.

16. A method according to claim 13, wherein the acoustic signal is a surface acoustic wave signal.

17. A method according to claim 16, wherein the acoustic signal is a travelling surface acoustic wave signal.

18. A method according to claim 16, wherein the acoustic signal is a standing surface acoustic wave signal.

19. A method according to claim 13, wherein an acoustic mismatch occurs at the interface of the plug and the carrier medium; and wherein the movement of the plug occurs because the interface of the plug and the carrier medium is displaced.

20. A method according to claim 13 wherein the at least one zone includes any one or more of:

(a) one or more reaction chambers;

(b) one or more outflow microfluidic channels;

21 . A method according to claims 14 or 15 wherein, the plug is controllably split into multiple daughter droplets by adjusting the input power of the signal source.

22. A method according to claims 14 or 15 wherein, at least one daughter droplet is directed into a zone and at least a second daughter droplet is directed into a second zone which is different from the first zone.

23. A method according to claims 14 or 15 wherein the daughter droplets are of uneven volume.

24. A method according to claim 13, wherein the carrier medium is a fluid.

25. A method according to claim 13, wherein the substrate is a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.