Patent Application
20160310947
The present invention relates to a method of controlling interactions between fluid compartments in a fluid flow. The present invention further relates to a method of generating a three-dimensional structure of vesicles or immiscible compartments. The present invention further relates to a method of controlling interactions between portions of a fluid in flow.
According to one aspect of the invention, there is provided a method of controlling interactions between fluid compartments in a fluid flow, comprising: providing a first phase within a fluid conduit; enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; providing at least one reagent compartment of a third phase that is immiscible with the second phase; and arranging the compartments such that at least one of the separating compartment or the at least one reagent compartment has a length that is equal to or greater than the fluid conduit diameter.
According to another aspect of the invention, there is provided a method of controlling interactions between fluid compartments in a fluid flow, comprising: providing a first phase within a fluid conduit; enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; providing at least one reagent compartment of a third phase that is immiscible with the second phase; and arranging the compartments such that at least one of the separating compartment and the at least one reagent compartment has a length that is equal to or greater than the conduit diameter.
Preferably, there are at least two reagent compartments. Preferably, the method further comprises arranging the compartments such that a thin film of the first phase is formed between the fluid conduit and both the separating compartment and the at least one reagent compartment.
Preferably, the method further comprises arranging the compartments such that a thin film of the second phase is formed between the at least one reagent compartment and the first phase.
Preferably, the method further comprises arranging the compartments such that the separating compartment and the at least one reagent compartment have different speeds of travel in the fluid flow. Preferably, the method further comprises arranging the compartments with predetermined spacings relative to one another in the direction of flow in the fluid conduit such that after a predetermined period of flow of the compartments the separating compartment confines the at least one reagent compartment to travel at the same speed in the flow. Preferably, the method further comprises arranging the compartments such that after a further predetermined period of flow of the compartments a further reagent compartment catches up with the confined reagent compartment due to travel at different speeds in the flow and interacts with the confined reagent compartment.
By arranging the compartments with predetermined spacings as recited above, migration and interaction of the compartments (also referred to herein as ‘droplets’) can be controlled, and in particular migration and interaction of the different reagent compartments e.g. mixing or being in contact for diffusion. One or more thin films may be formed between the fluid conduit and the reagent compartment.
As referred to herein, a ‘compartment’ may comprise a solid, a fluid or a combination of both a solid and a fluid. For example, a compartment (comprising a fluid) may be solidified within the fluid conduit, such as hydrogels/agarose solutions by temperature/UV control, for example, for the encapsulation of other media, such as cells, particles, DNA, etc. Thus, a compartment may be a fluid, a solid, contain solid particles, and/or may be turned from a liquid to a solid (and vice versa). In this context ‘confines’ preferably means causing the reagent compartment to travel at the same speed in the flow as the separating compartment. As used herein the term ‘thin film’ includes any form of film, of whatever thickness, but in particular a film sufficiently thin that a surface or interface affects the behaviour of the fluid; typically the thickness would be from micrometres to millimetres; preferably, a film is considered thin if it is less than 20% of the channel diameter (i.e. film thickness divided by tube radius 0.2). As used herein the term ‘immiscible’ preferably means fluids that separate over time if initially mixed and/or fluids that when placed in contact with each other do not substantially diffuse into each other. Reagent compartments may be miscible with one another, as they are of the same phase. A fluid conduit may for example be a channel, preferably a micro channel, a capillary or a tube, or a capillary/tube within a larger capillary/tube. As used herein, the term ‘phase’ preferably means a substance (whether a liquid, solid or gas) that is distinct from another substance, rather than a specific phase of a substance (e.g. liquid or gas). For example, herein, a fluorocarbon and water may be described as different phases. However, liquids and solids may also be referred to as (different) phases, in the appropriate context. As referred to herein, the term “fluid” preferably means a liquid and/or gas.
Preferably each of the reagent compartments has different compositions. Preferably, each of the reagent compartments may have different temperatures. Preferably each reagent compartment comprises a reagent. Preferably, the reagent compartments have properties such that when the reagent compartments are in contact with one another they merge and mixing of the reagent compartments occurs.
Alternatively, the reagent compartments may have properties such that when reagent compartments are contact with one another they do not merge and diffusion between the reagent compartments occurs. This can enable controlled diffusion between the different reagent compartments. Preferably at least one reagent compartment further comprises means for preventing merging. Preferably the means for preventing merging comprises a surfactant and preferably a lipid bilayer.
The method may further comprise selecting the properties of the separation compartment such that it moves in the fluid conduit slower than the reagent compartment(s) and arranging the separating compartment downstream of the reagent compartments. The method may further comprise selecting the properties of the or a further separating compartment such that it moves in the fluid conduit faster than the reagent compartments and arranging the or the further separating compartment upstream of the different reagent compartments.
The method may further comprise arranging the reagent compartments such that the first phase directly encloses the reagent compartments. The method may further comprise arranging the reagent compartments such that the separating compartment directly encloses the reagent compartments. If a reagent compartment is engulfed by a separating compartment, the reagent compartment will travel faster than the separating compartment, at least until such time as it reaches the interface between the separating compartment and the first phase, at which point its travel is ‘confined’ to the same speed as the separating compartment.
Preferably, the method further comprises selecting the properties of the three phases so that the surface tensions between the three phases are such that on contact between them the first phase encloses the second phase and the second phase encloses the third phase. Preferably, the length of the reagent compartment(s) (preferably individually and/or when enclosed) is equal to or greater than the conduit diameter. Preferably, the length of the separating compartment(s) is equal to or greater than the conduit diameter. The enclosing may occur spontaneously.
The method may further comprise providing at least two separating compartments, enclosing within the first phase a super-separating compartment of a fourth phase that is immiscible with both the first phase and the second phase, and arranging the compartments with predetermined spacings relative to one another in direction of flow in the fluid conduit such that after a predetermined period of flow of the compartments the super-separating compartment confines at least one of the separating compartments.
The method may further comprise arranging within the separating compartment an indexing compartment of a further phase that is immiscible with the second phase, and preferably immiscible with the third phase, and selecting the properties of the second, third and further phases so that the surface tensions between the three phases are such that on contact between them the second phase encloses the further phase, and the third phase and the further phase do not enclose one another.
Preferably, one or more indexing compartments are arranged between reagent compartments such that merging of reagent compartments is prevented. Each separating compartment may comprise an indexing compartment that has specific identifying properties for that separating compartment. The specific identifying properties are preferably compartment volume, compartment composition, or number of sub-compartments.
The method may further comprise flowing the compartments and then reversing the flow direction such that reagent compartments not confined by the separating compartment return to the arrangement with the predetermined spacings, or preferably to a predetermined spaced arrangement. The method may further comprise flowing the compartments in a flow direction and then reversing the flow direction such that a portion of the separating compartment breaks away.
Preferably the method further comprises aspiration of the different phases into a channel in a predetermined sequence to create the compartments with the predetermined spacings. Preferably the channel has only one inlet for aspiration. Preferably, the length of a compartment and/or the spacings between compartments in the fluid conduit can be determined by the duration for which a phase is aspirated.
As referred to herein, aspiration of a fluid into a channel or fluid conduit preferably means aspiration of a liquid and/or a gas—both of which are commonly understood to be fluids.
Preferably, magnetic particles may be used to transport media within and/or between reagent compartments during fluid flow, wherein the magnetic particles may be held in a fixed position by a magnetic field while the reagent compartments flow past. Preferably, the method further comprises using magnetic particles to transport media within and/or between reagent compartments during fluid flow, wherein a magnetic field may be used to move the magnetic particles within and/or between reagent compartments.
Preferably, the method further comprises transporting the magnetic particles between reagent compartments through the separating compartment. Preferably, the method further comprises transporting the magnetic particles between reagent compartments through the first phase. Preferably, the method further comprises transporting the magnetic particles between reagent compartments via a thin film that fluidly connects the reagent compartments.
According to another aspect of the invention, there is provided a method of creating a multiphase system comprising engulfing one or more separate fluid compartments by a second fluid, and engulfing that second fluid by a third fluid (optionally engulfing that third fluid by one or more further fluids) within a channel using interfacial tension between fluids to create such flow systems. Preferably the system is created in a channel with only one inlet. Preferably the length of individual compartments in the flow direction is equal to or greater than the channel diameter.
According to a further aspect of the invention, there is provided a method of controlling interactions between fluid compartments comprising selecting the properties (in particular the interfacial tensions) of three phase fluid compartments, so as to control interaction (and in particular relative speed of travel in a pressure driven flow) of the compartments.
According to a yet further aspect of the invention, there is provided a method of generating a three-dimensional structure of vesicles or immiscible compartments comprising: generating an interface between two immiscible phases in a vessel; and dispensing vesicles or immiscible compartments relative to the interface in order to dispense vesicles or immiscible compartments onto the interface.
Preferably, the method further comprises changing the properties of the two immiscible phases such that the vesicles or immiscible compartments transfer through the interface to the other phase.
Preferably, the method further comprises positioning a channel outlet for dispensing vesicles or immiscible compartments. Preferably the immiscible compartments are immiscible with either of the immiscible phases in the vessel. Preferably the vesicles or compartments are produced by any of the methods described herein.
Preferably, the method further comprises selecting the properties of the second phase and/or the third phase such that at a predetermined flow rate instabilities occur at an interface between the second phase and the third phase causing small emulsion compartments of the reagent compartment to be shed into the separating compartment.
Preferably, the method further comprises adding a surfactant to the separating compartment such that the interfacial tension is lowered between the second phase and the third phase.
Preferably the method further comprises arranging the compartments initially with predetermined spacings relative to one another in the direction of the fluid flow in the fluid conduit such that after a predetermined period of flow of the compartments the separating compartment comes into contact with the reagent compartment.
Preferably the method further comprises providing within the first phase in the fluid conduit a further compartment of a further phase that is immiscible with the first phase and second phase, arranging the further compartment downstream of the reagent compartment, and selecting the properties of the further compartment such that it travels in the fluid flow slower than the reagent compartment.
Preferably the method further comprises arranging the further phase to wet the walls of the fluid conduit.
According to a yet further aspect of the invention, there is provided apparatus for controlling interactions between fluid compartments in a fluid flow, comprising means for providing a first phase within a fluid conduit; means for enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; means for providing at least one reagent compartment of a third phase that is immiscible with the second phase; and means for arranging the compartments such that the length of the separating compartment is equal to or greater than the conduit diameter.
According to yet another aspect of the invention, there is provided apparatus for controlling interactions between fluid compartments in a fluid flow, comprising means for providing a first phase within a fluid conduit; means for enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; means for providing at least one reagent compartment of a third phase that is immiscible with the second phase; and means for arranging the compartments such that at least one of the separating compartment and the at least one reagent compartment has a length equal to or greater than the fluid conduit diameter.
Preferably, the apparatus comprises a fluid conduit. Preferably, the apparatus further comprises means for arranging the compartments such that a thin film of the first phase is formed between the fluid conduit and both the separating compartment and the at least one reagent compartment.
Preferably, the apparatus further comprises means for arranging the compartments such that a further thin film of the separating phase is formed between the reagent and separating phase, and such that the separating compartment and the reagent compartments have different speeds of travel in the fluid flow. Preferably, the apparatus further comprises means for arranging the compartments with predetermined spacings relative to one another in the direction of flow in the fluid conduit such that after a predetermined period of flow of the compartments the separating compartment confines at least one of the reagent compartments to travel at the same speed in the flow. Preferably, the apparatus further comprises means for arranging the compartments such that after a further predetermined period of flow of the compartments a further of the reagent compartments catches up with the confined reagent compartment due to travel at different speeds in the flow and interacts with the confined reagent compartment.
Preferably, the apparatus further comprises means for selecting the properties of the second phase and/or the third phase such that at a predetermined flow rate instabilities occur at an interface between the second phase and the third phase causing small emulsion compartments of the reagent compartment to be shed into the separating compartment.
Preferably, the apparatus further comprises means for adding a surfactant to the first fluid compartment such that the interfacial tension is lowered between the second phase and the third phase.
According to a yet further aspect of the invention, there is provided apparatus for generating a three-dimensional structure of vesicles or immiscible compartments comprising: means for generating an interface between two immiscible phases in a vessel; and means for dispensing vesicles or immiscible compartments relative to the interface in order to dispense vesicles or immiscible compartments onto the interface.
Preferably, the apparatus further comprises means for changing the properties of the two immiscible phases such that the vesicles or immiscible compartments transfer through the interface to the other phase. Preferably, the vesicles or compartments may be produced using any of the apparatus described herein.
According to a yet further aspect of the invention, there is provided a method of controlling interactions between portions of a fluid in flow comprising providing the fluid having a first phase within a fluid conduit; enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; arranging the first phase such that it has different properties downstream and upstream of the separating compartment.
Preferably, the method further comprises arranging the separating compartment such that the length of the separating compartment is equal to or greater than the fluid conduit diameter.
Preferably, selecting the properties of the separating compartment such that a thin film of a predetermined thickness of the first phase is formed between the fluid conduit and the separating compartment such that exchange between downstream and upstream of the separating compartment occurs at a predetermined rate.
Preferably, the method further comprises flowing the phases to support the exchange.
Preferably the different properties are different composition. Alternatively, the different properties may be different temperatures. Preferably the downstream composition comprises a mixture of differently sized components such that selective exchange of the components between downstream and upstream of the separating compartment depends on the thickness of the thin film.
The downstream composition may comprise a substrate and the upstream composition may comprise a component consuming the substrate. The substrate may be a reagent and the component consuming the reagent may be reacting with the reagent.
The rate of exchange of the substrate between downstream and upstream of the separating compartment preferably matches the rate of substrate consumption by the consuming component upstream of the separating compartment.
Preferably the consuming component is a biological organism. Alternatively, the consuming component may be a reagent or a catalyst.
The downstream composition may comprise a mixture of differently sized components and the thin film is such that selective exchange depending on size occurs. The downstream composition may comprise a mixture and the upstream composition comprises an environment that is selectively compatible with selective components of the mixture.
Preferably the method further comprises providing within the fluid conduit a further phase that is immiscible with the first phase and arranging the further phase such that it directly encloses the first phase.
The method may further comprise enclosing within the first phase a further separating compartment of the second phase or a third phase that is also immiscible with the first phase; arranging the further separating compartment upstream of said separating compartment; and selecting the properties of the further separating compartment such that a thin film of a predetermined thickness of the first phase is formed between the fluid conduit and the further separating compartment such that exchange between upstream and downstream of the further separating compartment occurs at a predetermined rate.
Preferably, the method further comprises selecting the properties of the or a further separation compartment such that it moves in the fluid conduit faster than the reagent compartments and the or the further separating compartment are arranged upstream of the different reagent compartments.
Preferably, the method further comprises providing an electric voltage between a first portion of the first phase upstream of the separating compartment and a first portion of the first phase downstream of the separating compartment for electrophoresis.
The method may further comprise fixing a substrate to the fluid conduit. Preferably the fixing comprises immobilising one or more magnetic particles by means of a magnetic field.
Magnetic particles may be used to transport media within and/or between reagent compartments during fluid flow, wherein the magnetic particles are held in a fixed position by a magnetic field while the reagent compartments flow past. Magnetic particles may also be used to transport media within and/or between reagent compartments during fluid flow, wherein a magnetic field is used to move the magnetic particles within and/or between reagent compartments.
Preferably, the second and/or third phase may comprise a fluid and/or a solid. Preferably, the method may further comprise solidifying (at least partially) a liquid contained in a compartment within the fluid conduit. Preferably, the apparatus may further comprise means for solidifying (at least partially) a liquid contained in a compartment within the fluid conduit.
The methods described herein have numerous applications, for example:
According to a yet further aspect of the invention, there is provided apparatus for controlling interactions between portions of a fluid in flow comprising means for providing a fluid having a first phase within a fluid conduit; means for enclosing within the first phase a separating compartment of a second phase that is immiscible with the first phase; means for arranging the first phase such that it has different properties downstream and upstream of the separating compartment.
Preferably, the apparatus further comprises means for arranging the separating compartment such that the length of the separating compartment is equal to or greater than the fluid conduit diameter.
Preferably, the apparatus further comprises means for selecting the properties of the separating compartment such that a thin film of a predetermined thickness of the first phase is formed between the fluid conduit and the separating compartment such that exchange between downstream and upstream of the separating compartment occurs at a predetermined rate.
The invention extends to methods and/or apparatus substantially as herein described with reference to the accompanying drawings.
The invention also provides a computer program and a computer program product for carrying out any of the methods described herein and/or for embodying any of the apparatus features described herein, and a computer readable medium having stored thereon a program for carrying out any of the methods described herein and/or for embodying any of the apparatus features described herein.
The invention also provides a signal embodying a computer program for carrying out any of the methods described herein and/or for embodying any of the apparatus features described herein, a method of transmitting such a signal, and a computer product having an operating system which supports a computer program for carrying out any of the methods described herein and/or for embodying any of the apparatus features described herein.
Any apparatus feature as described herein may also be provided as a method or use feature, and vice versa. As used herein, means plus function features may be expressed alternatively in terms of their corresponding structure, such as a suitably programmed processor and associated memory.
Any feature in one aspect of the invention may be applied to other aspects of the invention, in any appropriate combination. In particular, method aspects may be applied to apparatus aspects, and vice versa. Furthermore, any, some and/or all features in one aspect can be applied to any, some and/or all features in any other aspect, in any appropriate combination.
It should also be appreciated that particular combinations of the various features described and defined in any aspects of the invention can be implemented and/or supplied and/or used independently.
Furthermore, features implemented in hardware may generally be implemented in software, and vice versa. Any reference to software and hardware features herein should be construed accordingly.
These and other aspects of the present invention will become apparent from the following exemplary embodiments that are described with reference to the following figures in which:
Many bio/chemical reactions require several different constituents to be mixed prior to the reactions taking place, sometimes in a specific order, such as the Polymerase Chain Reaction for amplification of DNA. In addition there is often the need for multistep reactions that require a series of reactions to occur in a specific sequence, and at specific times, to achieve the required final product. A substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs, is often referred to as a ‘reagent’.
Currently employed technologies typically achieve such reactions by pipetting the required molecules at the required times, which can be labour intensive and prone to human error. Further application where control of exposure to reagents is crucial include probing cell-cell interaction, probing multi-cellular interactions, screening toxicity, materials development, microbiology, biological analysis, DNA studies etc. Controlled diffusion of different constituents in close contact with each other is another requirement for certain assays, for example for protein crystallisation or for cell-cell interactions.
A compartment (or ‘droplet’) containing a reagent may be referred to as a ‘reagent compartment’ (sometimes also referred to as a ‘sample’). In microfluidics, reagent compartments typically have for example picolitre (ph to microlitre (ml) volumes.
State of the art microfluidics allows controlled mixing/reactions on chips by bringing several different mixtures together at the required time, commonly referred to as a “lab on a chip” device or system. However, control of such systems is difficult to achieve due to the need for external sources to cause the coalescence of different reagent compartments, e.g. lasers, electrodes or complex channel networks of varying geometries that must be timed accurately to allow the coalescence at the required times. In such microfluidics networks many external sources are required for reaction timing and long start up times are often required to achieve a steady state operation.
Typically each lab on a chip design is for a specific experiment and there is little flexibility for using different reagents, number of samples, cells, number of reagent compartment merging events, reagent compartment spacing etc. Therefore each experiment requires the design of a new lab on a chip which is both time consuming and costly.
In known microfluidic mixing/reactions on chip it is typically a requirement to have surfactants as part of the chemical reaction to control the coalescence, although such surfactants can negatively impact the efficiency/accuracy and purity of the results. Uncontrolled coalescence can occur between sequential reagent compartments and render the results of any experiments/synthesis invalid, and the number of independent reaction steps that can take place is limited by micro-channel complexity, available size and excessive requirements for control.
In addition current lab on chip technologies are typically limited to several material choices with polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA) and polycarbonates (PC) being the most common, which also limits the fluids/materials that can be used within them. Chemical reactions can require aggressive fluids, which may not be compatible with usual microfluidic chip materials. For example PDMS is a commonly employed material despite having well-documented swelling issues with commonly used fluids.
Hence there is a need for a microfluidic technology that allows the controlled exposure to, and merging of, different solutions for control of the interaction of constituent parts.
The limitations of existing laboratory and lab on a chip based technologies can be overcome with a simpler, more efficient methodology of mixing reagent compartments in a controlled manner in a single channel with high resolution on both spatial and temporal parameters in a passive way. Also, the spacing between reagent compartments can be controlled for optimum heat and mass transport properties between walls or sequential samples, which has applications in heat and mass transfer areas of engineering science, for example. Thus, fluid physics in microfluidics are used to achieve mixing, separation and mass transfer, as opposed to geometry-based mixing as conventionally known in microfluidics.
In addition several external processes may take place such as heating/cooling, mixing, mass transfer etc. Gravity can be used as the pumping mechanism and thereby external pumps and electrical sources are unnecessary. Microfluidic multi-channel systems can be used. Alternatively external pumps (such as a syringe pump) can be used, to allow controlled mixing/reactions of reagents. Single or multistep reactions can be performed. The use of surfactants is optional.
The ability to manipulate micro- to femto-litre volumes has many practical applications, but current microfluidic devices are usually complex and/or dedicated to one purpose. Thus, the invention provides a simple solution that can be used by any experimental laboratory—a fluid conduit (such as a Teflon® tube) attached to a syringe pump. Flow through the tube is driven by gravity or a syringe pump, and interfacial tension and fluid mechanics are exploited to generate and merge any number of drops, and transfer precise volumes between them. More specifically, drops-within-drops are formed within the tube from three or more immiscible phases, and knowledge of tube diameter, interfacial tension, and flow rate is used to control when and where particular drops merge, and components are transferred between them. The invention can be used, for example, to create precisely-defined arrays (emulsions) of aqueous “cells” separated by lipid bilayers, crystallize proteins after serial dilution, ligate and amplify DNA sequences (perform the polymerase chain reaction (PCR)), and screen drugs for effects on human cells.
As will be described in more detail further on, the fluidic architectures described herein are preferably created at the inlet of a fluid conduit (or ‘channel/tube’) by an appropriate dipping between reservoirs and then as the fluidic architecture flows in the conduit the drops come together and merge.
A method for mixing and enabling multi-step reactions controls the relative position of samples in a channel, both spatially and temporally, thus allowing mixing of samples in a controlled way. At least three immiscible phases have different properties such that different fluid compartments travel at different speeds in a pressure-driven flow within a micro channel or capillary. The channel typically has a uniform diameter along its entire length, although a channel with varying diameter may also be used. However, a channel having a uniform diameter has a very simple geometry that is readily available in a range of materials and sizes and can be formed of a bio-compatible tube, for example.
As a first phase (also referred to as the ‘carrier fluid’) moves through the channel it wets the surface of the channel. A second immiscible phase (e.g. a reagent) forms a compartment (or droplet) which flows through the channel without making contact with the solid channel wall. The compartment is typically at least one channel diameter in length. The region of carrier fluid between the channel wall and the second phase compartment is termed the film region.
The film region can prevent contact between the second phase (and for example samples contained in the second phase) and the channel wall. Hence the same channel can be used to process multiple independent samples without possibility of cross contamination, and the same channel can be reused for independent experiments.
The thickness of the film region, also referred to as the film thickness, is determined by the carrier fluid dynamic viscosity (μ), velocity (V), interfacial tension (γ), length, surfactants and the viscosity ratio. The dimensionless capillary number
can provide a measure of the film thickness: the film thickness scales with Ca2/3. Bretherton's lubrication film theory can be used to evaluate the film region.
For the pressure-driven flow the velocity profile in the channel is parabolic (due to the no-slip boundary condition at the channel wall resulting in frictional drag). Compartments with a large film thickness (and consequently lying more centrally in the channel) occupy a faster portion of the channel, and consequently move faster than the average flow. Different compartments with different properties can have different film thicknesses, and therefore move at different relative velocities through a channel.
Selecting properties of the different compartments appropriately can enable the different compartments to move at different velocities in the channel, and hence a first compartment can catch up to a second compartment. The two compartments can then merge, allowing samples to mix.
In an example a system has a fluorocarbon as carrier fluid. Three different water-based compartments are formed in the carrier fluid. Each compartment has approximately the same density, but the interfacial tension is varied between compartments with different surfactant additives (e.g. different concentrations of TWEEN® 20—or other non-ionic surfactant that are particularly suitable for use in aqueous biological fluids) to the compartments. An upstream compartment has a low surfactant concentration and high interfacial tension, and hence forms a relatively thick film region and travels at relatively high speed in the pressure-driven carrier fluid flow. A downstream compartment has a high surfactant concentration and low interfacial tension, and hence forms a relatively thin film region and travels at relatively low speed in the pressure-driven carrier fluid flow. An intermediate compartment located between the upstream and downstream compartments has an intermediate surfactant concentration and intermediate interfacial tension, and hence forms a film region of intermediate thickness and travels at an intermediate speed in the pressure-driven carrier fluid flow.
Due to the difference in travel speeds the upstream compartment catches up with the intermediate compartment, merges and mixes, and then the merged compartment catches up with the downstream compartment, merges and mixes. In this manner, by selecting the compartments' properties, controlled mixing and reaction initiation can be achieved. Provided the initial spacing between the compartments is controlled, as well as the pressure driven flow, the times at which the reagent compartments merge can be controlled, in this example by selection of the interfacial tension of the compartments. The merging occurs passively requiring no external activation. By such controlled merging of compartments the contents of compartments can be varied discretely in time. The duration before compartments merge can be controlled in the range of seconds to days by appropriate selection of parameters. The time and position of a merging event is determined by user-selectable parameters including interfacial tensions, tube diameter, inter-compartment spacing, and flow rate.
External conditions required for chemical and biological reactions, e.g. thermal steps, gas diffusion, fluorescent recording/detection etc. can be applied through the correct choice of immiscible fluids/channel.
In another use of the above-described behaviour a sample or a number of samples (in separate compartments or dissolved in the carrier fluid) are collectively preceded by a first immiscible compartment, and followed by a second immiscible compartment. The first and second immiscible compartments have different viscosity ratios and/or capillary numbers, Ca, such that in a pressure-driven flow the rear immiscible compartment moves at a faster rate than the leading immiscible compartment and hence allow the samples to be brought into contact with each other at a controlled pressure where the force may be controlled by the two immiscible compartments. By thus bringing compartments into contact with one another the contents of compartments can be varied continuously in time. Continuous mixing within flowing reagent compartments would also be an advantage but can be suppressed by the use of surfactants through balancing the Marangoni effect and shear stress forces at the interfaces if desired.
In another use of the above-described behaviour an appropriate surfactant allows formation of a double micelle barrier where reagent compartments come in contact with each other but do not merge together. Instead of merging, the two compartments continue adjacent one another. Controlled diffusion can occur between the compartments across the double micelle barrier, in a similar way to lipid bilayers in real cell environments.
The approach described above requires accurate control of interfacial tension, which may not always be possible. In some situations mixing/reactions/diffusion may not occur in the desired sequence or at the correct rates or times. Another consideration is that for different samples (e.g. different aqueous based samples) the capillary numbers may be close to each other, in which case the relative speeds of travel are small and a long length of channel and/or a high pressure-driven carrier fluid flow velocity may be required for merging to occur.
To provide a better method of controlling the interaction of isolated compartments in microfluidics, a three phase system may be used. The three phase system can enable better control of merging and mass transfer between compartments, as is described below.
A number of reagent compartments 10-110-210-310-4 of the first phase are contained within a single separating compartment 12 of the second phase. The different reagent compartments 10-110-210-310-4 are miscible and therefore collectively considered to be of the same phase 10, but their composition varies from reagent compartment to reagent compartment for different reagents. The reagent compartments 10 occupy the central region of the channel 20, and therefore move faster than the separating compartment 12, as described above.
In the example illustrated in
By selecting the spacing between the reagent compartments 10 and the pressure driven flow in the channel 20 the times at which each of the reagent compartments 10 merges with the others can be controlled.
As described above, merging of the reagent compartments 10 can be suppressed by using an appropriate surfactant of suitable quantity to create a surfactant/lipid bilayer barrier between the reagent compartments 10, in which case controlled diffusion occurs when the reagent compartments 10 come into contact with one another.
In the system shown in
The carrier/separating fluid film region 18 can ensure that the reagent compartments 10 (containing for example different patient samples) never come in contact with the wall 20 and hence the same channel can be used to process multiple independent samples without possibility of cross contamination between separating compartments 12.
The separating/reagent fluid film region 19 between the reagents 10 and the separating compartment 12 is created as the fluid phase that forms the separating compartment 12 behaves like a solid wall relative to the reagent compartments. Therefore, as described above, each reagent compartment 10 within the separating compartment 12 moves at a higher velocity than the interface and therefore catches up with the next reagent compartment and merges if required.
Alternatively the reagent compartments 10 each have different properties with respect to their behaviour in the pressure driven flow. As set out above, the speed at which the individual reagent compartments 10 travel can be controlled for example by changing the interfacial tensions. Different reagent compartments 10 with different capillary numbers move at different relative velocities through separating compartment 12 in accordance with their film thickness, length, surfactant at interface and viscosity ratio. Therefore in addition to selecting the spacing between the reagent compartments 10, selection of the properties of each reagent compartment 10 can control merging and mixing of the reagent compartments 10 as they flow within the separating compartment 12. When all sample fluids are approximately the same density, the variation in interfacial tension between samples (either naturally or using surfactants/chemicals) can be controlled to initiate reactions in a controlled manner.
Alternatively surfactant concentrations can be controlled locally within the separating compartment 12 through either varying the concentration of surfactant between reagent compartments 10 or varying the surfactant concentration within the reagent compartment 10. Using this technique some reagent compartments 10 can be made to coalesce at the required time while others can be made to come into contact with each other and allow mass diffusion between them within the same separating fluid compartment 12. The rate of mass transfer between reagent compartments 10 can be controlled by varying the physical distance between reagent compartments 10 (or chemical concentration) within the separating fluid compartment 12, or varying the properties of the separating fluid itself such as surfactant concentration. The result is a number of independent separating compartments 12 within a channel which can allow communication between individual reagent compartments 10 within each separating compartment 12. By varying the properties of the carrier fluid 14 it is also possible to control the distance, merging and communication between the separating compartments 12.
As mentioned earlier, it should be noted that in other similar examples the reagent compartments 10 may comprise a solid phase (or may partially contain a solid).
Provided the volume of the compartments is small enough that the effect of gravity on the compartments is negligible, the interaction between compartments is based only on interfacial tension effects, and consequently the volume of the compartments has no effect on the behaviour, and the compartments can be controlled in the same manner independent of the compartment size. This has been demonstrated with droplet volumes ranging from ˜1 nl to ˜2 μl respectively in tubes of diameter 50 μm to 620 μm.
Changing the flow direction allows the reagent compartments to return to their original position and spacing between them as long as no reagent compartment has reached the front of the separating compartment. Such systems can be made parallel by adding multiple channel systems. High throughput can be achieved both by loading multiple separating compartment in sequence and also by adding additional channels in parallel. Tens of channels can be operated in parallel with a single pump, in particular in the case of channels formed of simple tubes.
Each reagent compartment can be recovered at the channel outlet and detailed analysis performed using conventional techniques. Further manipulation of a droplet outside of the tube is possible, as is long term storage of droplets for example in well plates.
The creation of double emulsions containing reagent compartments has been demonstrated in microfluidic devices using flow-focussing junctions (with a variety of fluids, hydrophobic and hydrophilic surfaces). Due to the method of manufacture the emulsion is however typically limited to a single reagent compartment per separating compartment. If the described devices were adapted to form multiple reagent compartments per separating compartment, then due to the method of manufacture the reagent compartments would necessarily all be of the same composition.
Formation of double emulsions containing multiple reagent compartments has been demonstrated in microfluidic devices with co-axial flows combined with the intelligent use of surfactants. In the co-axial flow device multiple reagent compartments within the same separating compartment are possible, but due to the method of manufacture the reagent compartments are necessarily all of the same composition. In order to prevent merging of sequential reagent compartments only negligible fluid property variations are possible.
None of these known methods of producing double emulsions in microfluidic devices provides control of the spacing between the reagent compartments, or indeed of the positioning and interaction between the reagent compartments within the separating compartment, nor would this be desirable as it would cause the already complex channel networks required to be even more complex.
In known double emulsion devices the droplet generated (both the reagent compartments and also the separating compartment) is of smaller length in the flow direction than the channel width. The reagent compartments are not made to travel in a channel that constricts them into elongate compartments, nor would this be desirable as it would cause the laboriously produced reagent compartments—of the same composition—to merge, to no particular purpose. Unlike conventional three phase microfluidic systems that generate spherical drops within a carrier spherical drop, the described three phase system functions on the basis of a slug flow, where the length of slugs is at least the dimension of the channel geometry, under which condition thin film formation occurs. The length in the flow direction is equal to or greater than the channel diameter for the relative motion to be controllable.
Instead of using multiple reservoir aspiration to produce the three phase system as described above, a conventional device can be adapted to combine first a microfluidic/microchannel network to produce a variety of different reagent compartments in a particular arrangement (controlling both spacing and sequence of the reagent compartments), and then feed the reagent compartments into a microfluidic system for generating an emulsion. This would require a complex channel network with sophisticated control of a number of auxiliary equipment, and is therefore larger and more cumbersome, expensive, difficult to operate, and more prone to error and failure.
In order to ensure the different compartments are appropriately positioned in the channel, an aspiration based method of forming the compartments is now described, which is suitable for the aspiration of both liquid and gas (i.e. fluids) in the present invention.
The three immiscible fluids are aspirated in the desired sequence into a channel. A simple one dimensional channel requiring no junctures, with a single inlet, is suitable for aspiration. To effect aspiration, that is an inflow of fluid into the channel opening, a hydrostatic pressure difference can drive the inflow with gravity as the driving force, or capillary effect can be employed to aspirate fluids, or external pumping or vacuum devices can be applied to cause a flow. The different fluids can be aspirated from different, separated reservoirs (e.g. by sequentially bringing the channel inlet into fluid communication with different fluid vessels). Alternatively, the different fluids can be aspirated from a single reservoir that contains different immiscible phases, as shown in
To generate the arrangement of compartments shown in
By selecting the amount of the second immiscible fluid aspirated 22-2 after each reagent compartment 10, the separation between the reagent compartments 10 can be controlled, and thus the duration of travel before mixing of the reagent compartments 10 occurs. The reagent compartments 10 need not be regularly spaced with equal intervals between them, as shown in
In addition to providing ease of control of the arrangement of different compartments in a channel, the aspiration based method of forming compartments requires only a single channel with only minimal external equipment and no particular channel geometries, thereby providing cost, size and usability advantages over other methods of forming the compartments.
The aspiration based method of forming compartments is not limited to four reagent compartments as shown in
The sequence of carrier fluid, reagent, separating fluid as described above can be repeatedly aspirated into a channel to create a number of independent reagent compartments engulfed by the separating compartments engulfed by the carrier fluid, with each containing the same/similar or unrelated constituents. In an example, each separation compartment contains first a sample that is being investigated, with a different sample for each separating compartment, and in each separation compartment there are three other reagent compartments that contain always the same three different reagents used in the analysis of the samples.
Now the interfacial tension requirements of the different phases are considered in more detail. For the interface between three immiscible phases to be in equilibrium requires that the Neumann triangle be satisfied. This can be stated as a requirement that the interfacial tension γ between any two fluids cannot be greater than the combination of the interfacial tensions between the other fluids and may be expressed as the following inequality
γ1-2<γ1-3+γ2-3
This inequality has been used to identify suitable spacers to prevent merging of water droplets in microfluidic devices. However, if γ1-2 is greater than the sum of γ1-3 and γ2-3, then fluid 3 forms the interface between fluids 1 and 2 which may be used to create an engulfing effect of one fluid on another.
The micrographs in
γFC40surfactant/water<γFC40+surfactant/silicone oil+γsilicone oil/water.
The three phases exist in equilibrium in a triple interface. The lower image shows the case (FC40 without surfactants) that does not satisfy the Neumann triangle:
γFC40/water>γFC40/silicone oil+γsilicone/water.
The silicone oil forms an interface between the water and the FC40, and in doing so engulfs the water plug. In the right of the image silicone oil breaking away from the water droplet is visible. The outcome is a water droplet engulfed by the silicone oil, which is in turn engulfed by the FC40.
In multi-phase microfluidic flow systems where the inequality of the Neumann triangle is not satisfied, by virtue of selection of suitable fluid properties and control thereof, sophisticated control of fluid interactions can be enabled.
Now an example of a system as described with reference to
A PTFE tube 20 filled with fluorocarbon FC40 14 has an alternating stream of water 10/silicone oil 12 droplets aspirated into it. The channel diameter is typically about 0.6 mm, but can be for example 2 orders of magnitude smaller or larger. The interfacial tensions between FC40/water, FC40/silicone oil and water/silicone oil are measured to be 44 mN/m, 4.3 mN/m and 25.9 mN/m respectively using the pendant drop technique. The result is a microfluidic system where the fluorocarbon FC40 14 wets the capillary wall 20 and engulfs the silicone oil 12, which in turn engulfs the water droplets 10 as shown schematically in
Now variants of the system according to
The channel walls 20 in the portion of the channel containing the gas phase 15 are dry; the separating fluid 12 wets the channel walls 20. In order to ensure the walls are sufficiently dry for the necessary interface to form, the channel needs to be sufficiently dry, and may not still be wetted from a foregoing fluid in the channel. A mere bubble of gas is not necessarily sufficient to form a suitable interface.
More specifically, the reagent compartments 10 cannot overtake the blocking compartment 32 and therefore it confines the first reagent compartment 10-1 that catches up with it and forces that reagent compartment 10-1 to travel at the same velocity. The subsequent reagent compartments 10-210-3 then in due course catch up with the first reagent compartment 10-1 and merge one by one, as illustrated in
Alternatively, the slow travelling compartment 30 and the single blocking compartment 32 could be combined as a single compartment selected such that it is immiscible with, and moves slower than, the reagent compartments 10. Again, such a slow-travelling blocking compartment (not shown) would perform a similar function to the gas/separating fluid interface 17 described with reference to
Also with reference to
In another alternative system to the three-phase system shown in
In an alternative, the fourth super-separating compartment 34 fluid and the reagent compartments 10 fluid can be miscible fluids, e.g. both aqueous based, separated by an immiscible fluid and hence transfer of material across the film region separating the two fluids can occur.
The reagent compartment 10-3 that is thus contained in the separating compartment 12 adapts by behaving as if the separating compartment 12 were a new solid boundary, and now occupies a narrower central region of the channel 20 than the separation compartment 12. As described above, the reagent compartment 10-3 cannot pass the interface 16 between the separation compartment 12 and the carrier fluid 14, and therefore remains constrained at the front of the separating compartment 12. The reagent compartment 10-3 and separating compartment 12 continue to travel at a greater velocity than the reagent compartments 10-110-2 alone since the reagent compartment 10-3 is now inside the separating compartment 12. One by one the reagent compartments 10-210-1 are engulfed from the rear by the separating compartment 12 in sequence of their arrangement in the channel. Each new reagent compartment 10-2 that is engulfed by the separation compartment 12 may merge with the reagent compartment 10-3 already inside the separating compartment 12.
Alternatively, merging of the reagent compartments 10 within the separation compartment 12 can be suppressed by using an appropriate surfactant of suitable quantity to create a surfactant/lipid bilayer barrier between the reagent compartments 10, in which case controlled diffusion occurs when the reagent compartments 10 come into contact with one another within the separation compartment 12.
Instead of or in addition to using the fourth immiscible fluid described above for separation of reagent compartments 10, one or more such compartments 36 is included in each separating compartment 12 for the purposes of indexing. This is particularly useful where a number of separating compartments 12-112-2 differ between one another, for example with each separating compartment 12 containing a particular patient sample. Especially if the separating compartments 12 are released from the channel 20 and the ordering of the separating compartments 12 changes, indexing of the individual separating compartments 12 in order to identify the particular content of the separating compartments 12 for post analysis. This can be particularly important for large numbers of different separating compartments 12. Such an indexing compartment 36 is contained within a separating compartment 12 and is of a fluid such that it does not merge with any of the reagent compartments 10. The indexing compartment 36 can enable identification on the basis of for example molecule free indexing (reagent compartment 10 size/volume) or composition (e.g. fluorescent gradient reagent compartments 10), or reagent compartment 10 number. Alternatively a number of indexing compartments 36 can combine to encode an identifier (e.g. a binary identifier). Indexing compartments 36 can be formed by aspirating a single or a number of indexing compartments 36 to each separating compartment 12 and thereby being able to identify the exact original constituents of each separating compartment 12. The Indexing compartments 36 can be, but do not need to be, immiscible with the reagent phase 10. For example the indexing can be an aqueous phase with varying fluorescent and size properties.
Alternatively, when the analysis is achieved entirely within a channel 20 and the arrangement of the different separating compartments 12-112-2 relative to one another is maintained, the indexing compartment 36 can be engulfed by the carrier fluid 14 instead of being contained within the separating compartments 12, as the association between indexing compartment 36 and separating compartment 12 is unambiguous.
Detachment occurs under suitable conditions with respect to interfacial tension forces and shear stress/drag forces (unlike engulfing, which is determined by interfacial tension alone).
Each reagent compartment delivered to the reservoir can have been formed from a number of reagent compartments merging during the journey to the channel outlet. In the example illustrated in
In an example each reagent compartment delivered to the reservoir contains different cells types expressing different molecules. In another example each compartment contains a number of sub-compartments, for example an indexing sub-compartment and a cell sub-compartment. The interaction between these separating compartments is probed by having the fluid properties of the buffer fluid in the reservoir porous to the molecules of interest. A porous buffer fluid is one that those molecules are soluble in, and/or diffusion of those molecules occurs in that buffer. All fluids are somewhat porous and can act as solvents under the right conditions. In the arrangement of the compartments in the buffer reservoir there is a layer of reservoir fluid between compartments and hence the reservoir fluid are soluble/porous to the molecules of interest. Therefore they must pass through the bilayer, then the reservoir fluid. For example silicone oil is highly porous to fluorescein however fluorocarbons are not porous to fluorescein.
In an alternative the buffer fluid in the reservoir could have properties that are non-porous to the molecules of interest and the separating compartments could be arranged in the desired 2D/3D structure, to represents a multicellular environment and/or with variation of gradients in molecules across the environment (e.g. reflecting a cancer lump where the outer cells have a high concentration of drugs while the inner ones have a lower concentration). When the desired arrangement is achieved, and required time has passed, the entire arrangement may be transferred to a buffer fluid which is porous to the molecules of interest, by varying the surface tension of the fluids interface. In this arrangement the non-porous/porous buffer fluid interface is such that it does not allow the separating compartments to pass through the interface, until the interface properties are modified and hence the separating compartments can transfer from the non-porous buffer solution to the porous buffer solution. This can be achieved through the addition of surfactant to the buffer region/s. The reverse, where the separating compartments move from the porous to the non-porous buffer fluid can be achieved using a similar methodology.
In another example the different compartments can be transferred from the channel outlet to either an unconfined surface or a reservoir to monitor the individual separating compartments over time. Evaporation of fluids form the compartments can be reduced or increased through either humidity control or choice of carrier and separating fluids. In particular, the different compartments can be transferred to standard biotechnology formats such as 96 well plate readers, PCR machines etc. for further analysis.
Each reagent compartment can be recovered and detailed analysis performed using conventional techniques with the aid of indexing of each separating compartment.
In all of systems described above the following operations and alternatives are possible:
In another example of an application of a dilution series for RT-PCR (reverse transcriptase PCR) amplification is prepared. Instead of preparing sample by conventional pipetting, the five required reagents (a buffer, primers, RT-Taq and DNA-Taq enzymes, and template) are loaded in six separating compartments. Each separating compartment contains five reagent compartments, one reagent compartment for each of the five required reagents, appropriately spaced and sequenced. From separating compartment to separating compartment the amount of template varies so as to produce the dilution series. As the flows along a tube the reagent compartments merge and mix to create six mixtures in a dilution series. At the tube outlet the resultant six mixtures are deposited in six different wells of a 96-well plate alongside their analogues prepared conventionally by pipetting. After amplification in a PCR cycler and gel electrophoresis, the dilution series prepared in the three phase system is observed to be identical to the dilution series prepared conventionally by pipetting. By producing the dilution series in a three phase system rather than by pipetting the biocompatibility risks of working with biological matter can be reduced, as the reagents are isolated (by the separating compartment, the carrier fluid and the tube) from the environment.
In the example of
In this example, instabilities are created at an interface 2014 between the reagent compartment 2010 and the separating compartment 12 so as to cause shedding of small emulsion compartments 2012 from the reagent compartment 2010 into the separation compartment 12. This can enable emulsification of a larger compartment 2010 into smaller compartments 2012. The individual emulsion compartments 2012 can be of volumes in the range of femtolitre to picolitre. The rate at which emulsion compartments 2012 are generated from the reagent compartment 2010 can be in the range of kHz. Instabilities that result in the shedding of the emulsion compartments 2012 may be created by adding suitable surfactants to the separating compartment 12, thereby lowering the interfacial tension with the reagent compartment 2010.
Here, the reagent compartment 2010 may be aqueous, for example, and is engulfed by a larger separating compartment 12 of the mineral-oil mix used for emulsion polymerase chain reaction (PCR), which is enclosed in FC40 carrier fluid 14. The resulting interfacial instability causes small aqueous droplets to be shed into the separating compartment 12. For example, a 20 nl reagent compartment 2010 provided in a 150 μm diameter tube may yield an emulsion in which aqueous emulsion components 2012 have volumes ranging from femtolitre (fl) to picolitre (pl). In an alternative, the PCR oil (which can permit permeation of small molecules) is replaced with mixtures known to be suitable for generating emulsions containing proteins (such as a mixture of tetradecane, EM180 and Span® 80); in this case emulsions are produced that both remain stable during thermal cycling and can retain fluorescent molecules for at least two weeks.
To generate an emulsion as described above only a single tube with a single inlet is required, without requiring a T-junction or other flow focusing device, a complex channel of networks with multiple syringe pumps controlling the flow, or any mechanical means such as agitation, as previously used.
The generated emulsion is contained with the separating compartment 12, which means that each aqueous reagent compartment 2010 and separating compartment 12 can act as an independent sample, free of contamination from other samples. With conventional emulsion generation such containment would require a complex setup including a number of parallel droplet generating devices with all associated auxiliary equipment and connectors.
Subsequent size filtering (for example as described above with reference to
In another example (not shown) a number of reagent compartments 2010 can be made to merge and react as described with reference to
When working with live media in microfluidics, currently it is not possible to continuously refresh the surrounds of such media. For example a cell culture in a reagent compartment consumes the nutrients supply and excretes waste products hence changing its environment with time.
For example, the carrier fluid 1100 is an aqueous phase (with the channel walls 20 suitably hydrophilic) and in one of the portions 1100-3 a cell culture is contained. The cell culture portion 1100-3 can have a continuous refreshment of its culture media supplied by an adjacent portion 1100-2 through the film region of a separating compartment (e.g. of silicone oil, fluorocarbon or air), while used medium waste can be extract from the cell culture portion 1100-3 at the same rate to another upstream portion 1100-4, thereby maintaining a constant internal environment in both size and chemical properties. Alternatively, the cell culture environment can be fed from several downstream portions 1100-11100-2 to allow a controlled variation of the cell culture portion 1100-3 environment for perturbation of the cell culture 1100-3 by a compound for screening purposes. Such systems enable maintaining a fixed cell culture environment and open up new potential in microbiology, toxicity and drug screening. This system can also be used to probe 3D cell culture for controlled diffusion between samples with different proteins/cell lines for example.
Alternatively, the cell culture portion may be fed from several downstream portions to allow a controlled variation of the cell culture environment for screening purposes. A range of different molecules can be arranged in different reagent portions to undertake screening with varying temporal concentrations of desired molecules. Because the reagent is completely contained within the carrier fluid and at no point directly contacts the channel walls, there is no risk of fouling of the channel (as can occur in the example described with reference to
The thickness of the film region 1108 between the separating compartments 1102 and the reagent compartments 1110, in effect the height of the pathway between reagent compartments 1110, is estimated as follows. The thickness of the film region 1108 relative to channel/tube 20 diameter is proportional to the Capillary number (Ca) to the power of ⅔. For tubes of 150-1000 μm inner diameter with average flow velocity of 0.25-6 mm/s the thickness of the film region 1108 is estimated in the range of 0.1-40 μm.
In a further example of a three phase system for particle sizing (not shown), fluids may be aspirated into a 150 μm PTFE tube in the following sequence (with the first being the leading compartment farthest downstream):
As the sequence of compartments flow in the tube, the aqueous fluid compartment and the filtered water compartment merge as an engulfing aqueous channel (thickness ˜3 μm) forms around the separating fluid. After the aqueous channel is formed the larger particles remain in the leading aqueous compartment, downstream of the separating compartment, while the smaller particles migrate and accumulate in the lagging aqueous compartment upstream of the separating compartment, because only the smaller particles are small enough to pass through the aqueous channels that connect the leading and lagging parts of the merged aqueous compartment.
In an alternative system, magnetic particles can be used to move media from one part of a merged aqueous compartment to another and/or or between discrete aqueous compartments through the separating fluid as the flow moves them past the particles. This can be achieved by using a magnetic field to hold the magnetic particles in a fixed position as the compartments flow past. It is also possible to use a magnetic field to move magnetic particles from one part of a merged aqueous compartment to another and/or between compartments. In a further alternative system a further compartment containing drugs/markers may be added to the second half of a merged compartment during diagnostic applications.
In this example, fifteen independent reagent compartments are arranged in a conduit (as shown centrally in the figure). The magnetic particles (‘beads’ in this example) are moved as follows:
This process is suitable for gene assembly with multiple wash steps.
With reference to
With reference to
In a similar three phase system to that shown in
In the system shown in
An example of a three phase system for mass transport is now described in more detail. For any such system, the fluids interfacial properties should satisfy the inequality of:
γcarrier/separating>γcarrier/reagent+γseparating/reagent
An example of fluids used in such a three phase system is:
In another example the reagent compartment is water without a surfactant. In this example, the surfactant in the carrier fluid is selected to achieve a sufficiently low interfacial tension between the carrier fluid and the reagent compartment. In the case of the carrier fluid being a fluorocarbon such as FC40, a suitable such surfactant is Pico-Surf, with others being available. The flow rates between neighbouring reagent compartments is calculated based on the difference in velocity of the leading and subsequent droplets and depends on the carrier fluid mean velocity.
{dot over (Q)}∝V1.57
Another parameter that can be varied for achieving a desired flow rate is the tube diameter D. Provided the capillary number and fluids remain same the flow rates between reagent compartments, {dot over (Q)}, scales with the tube diameter, D, as follows:
{dot over (Q)}∝D2
By suitable parameter selection femtolitre flow rates and lower can be selected.
The film thickness of the fluidic pathways may be estimated using the assumptions that μseparating>>μreagent (where μ is dynamic viscosity) and therefore the separating compartment behaves as a solid plug and hence the theory of M. E. Charles (Can. J. Chem. Eng. 41, 46 1963) is applicable providing a range of film thicknesses from ˜1-20 μm for the range of flow rate measurements presented in
By controlling the mean flow rate within the tube the size of the fluid pathways connecting adjacent reagent compartments can be selected. The mean flow rate within the tube can be controlled using either mechanical means or alternatively using gravity feed systems. Similarly, for particle size separation the film thickness can be controlled to a high degree of accuracy by an appropriate selection of separating compartments such that the film thickness between the separating compartments and the carrier fluid is suitable for particle size filtering.
In another variant of the three phase systems for mass transfer described above, two or more reagent compartments that are initially separate may be merged at a specific time. The merging can for example be achieved by way of a blocking compartment, in a similar manner as that described earlier with reference to
In a variant of a three phase system for mass transfer, described with reference to
More specifically,
Implementation of this Technology in the Field of Biomedicine
It will be understood that the present invention has been described above purely by way of example, and modifications of detail can be made within the scope of the invention.
Each feature disclosed in the description, and (where appropriate) the claims and drawings may be provided independently or in any appropriate combination.
Reference numerals appearing in the claims are by way of illustration only and shall have no limiting effect on the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1321433.3 | Dec 2013 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2014/053614 | 12/4/2014 | WO | 00 |
