Patent Application
20150174576
This invention relates to a microfluidic system for generating droplets and allows merging of droplets in a controlled and synchronised manner, in order to achieve an improved analytical device and methods.
There has been a continued desire for using smaller sample quantities, which has led to research being carried out into transferring many known analytical techniques to the sub-millimeter scale. Recent advances in microfluidic control architecture have significantly improved the ability to work with materials on a microscale, for example, allowing precise temporal and spatial manipulation of single droplets and functions such as sorting, splitting and merging droplets for complex analyses.
Droplet-based microfluidics has emerged as a valuable platform for performing high throughput chemical and biological experiments. In such systems, droplets are made to spontaneously form when laminar streams of aqueous reagents are injected into an immiscible carrier fluid, either at a T-junction or in a flow focusing geometry.
Biological processes in microdroplets have been described in Current Opinion in Chemical Biology, 2010, 14:1-8; Mol. BioSyst. 2009, 5, 1392-1404; and Lab Chip. 2008, 8, 1244-1254. Processes that have been carried out inside droplets include DNA amplification, protein expression, enzymatic turnover, cell growth and multi-cellular organism growth.
Known microfluidic devices are associated with various practical difficulties, including low versatility, high cost and sample cross-contamination.
US 2009/0288710 discloses a microfluidic sampling system for taking samples of fluid from at least 2 wells from above, using a microsampling head. A first well, contains, e.g., a sample covered by a second fluid (e.g. an oil) that is immiscible with the first fluid. A second well contains, e.g., a reagent, covered by a second fluid (e.g. an oil). The microsampling head draws in a volume of the sample from the first well, a volume of the second fluid of the first well, and is then moved to the second well to draw in a volume of the reagent of the second well. A third fluid, acting as a wash fluid, may cover both wells such that they are in fluid communication. This system addresses is cross contamination by virtue of the wash fluid, evaporation of the sample by virtue of the second fluid layer over the first fluid layer, and unwanted introduction of air by virtue of keeping the wells in fluid communication by means of the third fluid.
Series of alternating droplets are prepared by moving the microsampling head between different wells to draw in different samples interspersed by carrier fluid. Pairs of consecutive droplets (e.g. sample and reagent), separated from each other by the second fluid in the microsampling channel, may be combined by virtue of a downstream increase in the cross section of the microsampling channel, which is large enough so that the droplet of the second fluid is no longer able to fully separate the two consecutive droplets, which then mix.
A valve system is used to achieve droplet sampling and to conduct the droplets into the system. The valve is set to a first position, to pull a droplet out of the sampling reservoir. When the droplet passes the valve, the valve is switched, to push the droplet onwards through the valve and along the tubing into the downstream system.
There are various practical difficulties associated with this device, including contamination around the valve joint, the need to use large sampling volumes, and the slowness of the system, with a relatively low throughput. Data in US 2009/0288710 shows that samples are taken only once every 60 seconds. This indicates the large spacing present between sample droplets in the system.
Droplet dilution has been reported using a passive geometry in which a trapped mother droplets is diluted down by a series a dilution droplets. Other dilution mechanisms include the creation of a chemical gradient using diffusion and laminar flows before encapsulation in an oil phase. (Ref: Niu et al., Nature Chemistry (2011), 3; 437-442)
U.S. Pat. No. 7,129,091 discloses a microfluidic device for manipulating fluid plugs in channels. Both the merging of a plurality of fluid channels into a single fluid channel, and the splitting of fluid plugs into two or more channels is disclosed. Mixing of fluids by generating chaotic or turbulent flow by channel geometry is also disclosed.
‘Plugs’ of fluids containing different reagents can be merged by passing them through a T-shaped region in a channel and having the droplets meet head on or travel at different velocities. Pairs of plugs can be merged by controlling the arrival time of plugs flowing in opposite direction towards a common area, so that the plugs arrive ‘at around the same time’ to form a single plug. The sequential arrival of droplets into a merging chamber is also disclosed. The exact arrival time, and exact order of droplet arrival is said to be difficult to control.
US 2006/0094119 discloses a system for microfluidic manipulation of small volumes of solution. The system is configured into a loading component, a holding component, a combining component and a receiving component.
The detachable holding component may hold the droplets in a linear array. Two arrays of droplets can be stored such that they may be merged, and the merged plugs transferred to the combining component for further reactions. Each holding component includes a series of different reagents. The combining component can be used to merge plugs with other plugs, such as a stream of reagent plugs, for example, by injection.
The droplets may be transferred to a receiving component, such as a microchannel, e.g., a length of tubing. Reactions in plugs can be monitored in any of the components of the system or after exiting the system.
In the prior art systems it is very difficult to accurately control the merging of droplets. The present invention represents a significant improvement over the current state of the art in providing a simple device, able to generate droplets from very small sample volumes in a controlled manner, merge droplets in a is controlled manner, generate large amounts of very accurate data for chemical and biological studies, and operate at a low cost compared to the current standard devices.
The present invention relates to a microfluidic system for sequential sampling of droplets. The system comprises:
Optionally, the invention may further comprise a microfluidic chip supporting channels or capillaries to conduct one or more droplets in a carrier fluid. The tubing or capillary may connect the microsampling tip with the microfluidic chip.
Microfluidic chips suitable for the current invention are known in the art. They may be made of glass or polymer and fabricated using standard microfabrication techniques adapted from the semiconductor industry. Soft lithography in particular has been extensively used by the microfluidics community. See “Soft Lithography in Biology and Biochemistry”, Whitesides, G. M., Ostuni, E. S., Takayama, S., Jiang, X., and Ingber, D. E., Ann. Rev. Biomed. Eng, 2001, 3, 335-373. Suitable examples include glass or plastic chips from Dolomite Microfluidics, ChipShop, Micronit, Micralyne, ThinXXS, Ibidi. These chips include a variety of functions in either 2-phase or continuous flow.
The present invention relates to a droplet generator.
The device comprises an electronically driven microsampling head, which comprises an opening, or ‘microsampling tip’, for generating droplets of fluid. The opening (microsampling tip) is positioned under a container, reservoir or tank, loaded with at least two fluid phases, wherein adjacent phases are immiscible with each other. The microsampling tip is moveable relative to the container, reservoir or tank. The microsampling tip is optionally movable at least laterally and vertically. This can be achieved by electronic control and may be pre-programmed.
Fluid aspirated by, or pushed through, the microsampling tip passes to a microfluidic chip, via a section of tubing or a capillary connecting the microsampling tip with the microfluidic chip. Optionally, a connector means may be incorporated between the tubing or capillary and the microfluidic chip.
Pressure is applied to the system in order to achieve aspiration of droplets by the microsampling tip into the system. In one embodiment, negative pressure is applied to the system at the far end of the tubing or capillary system on the side of the microfluidic chip distant from the tip. When the microsampling tip is positioned in a fluid phase, the application of negative pressure causes fluid to be aspirated into the tip and flow into the tubing. In another embodiment, positive pressure is applied. In a further embodiment, positive and negative pressure are both applied to the system.
By moving the microsampling tip between a first fluid phase and a second fluid phase, and back into the first phase, droplets are aspirated and pulled into the tubing. Repeated moving of the tip between the first and second fluid phases aspirates a series of droplets of a first fluid phase, with each pair of droplets separated by a volume of the second fluid phase. The retention time of the microsampling tip in each phase, in conjunction with the size and shape of the tubing or capillary, determines the size of droplets generated and conducted into the tubing.
The system of the invention may be configured to be used as a droplet synchroniser.
In this embodiment, the droplet generator comprises at least two electronically driven openings (‘microsampling tips’), which aspirate droplets from at least one sample well. The number of openings (microsampling tips) corresponds to the number of samples. Each microsampling tip in the droplet synchroniser system is dedicated to aspirating droplets from only one type of sample. A tubing section is associated with each microsampling tip to allow parallel droplet generation. The generation of droplets occurs in the same manner as described above for the droplet generator system.
Droplets can be merged with each other on the microfluidic chip or in a capillary in a controlled fashion. Various merging methods are possible and envisaged by the present invention. For example, on-chip or in capillary merging allows for controlled merging of two or more sequential droplets, and also allows for one or more droplets to be merged with one or more droplets generated in parallel by different microsampling tips. Further, it is also possible to control the merging of a droplet from a first sample well and a droplet from a second sample well into one combined droplet.
Pairs, triplets, quadruplets etc. of droplets are generated in sequence in a capillary using the described robotic head. The relative distances between droplets will depend on capillary geometry, droplet size, speed, viscosity and interfacial tension between the oil and droplet. By tuning these parameters, merging can be obtained in a controlled manner and get the pairs, triplets, quadruplets etc. of droplets to merge after travelling a desired distance within the capillary.
Droplets can be delivered into a capillary and merged in a controlled fashion. For example, a first droplet can be generated from a first sample, and a second droplet can be generated from a second sample—both generated at low flow rate. The droplet can merge with any of the available sample fluid, including the first or second sample, again to generate a third, fourth or fifth droplet. The droplets can then be merged into one or more combined droplets in a controlled fashion directly in capillary. This merging can, for example, be achieved by subsequently increasing the flow rate. Combinatorial mixing and dilution gradient can thus be achieved.
Droplets can be delivered to one location or multiple locations on the microfluidic chip, in a controlled fashion. For example, droplets delivered to the microfluidic chip from a first tubing, associated with a first sampling fluid, can be delivered to a channel on the microfluidic chip at a location that is the same as, or different to, the location at which droplets from a second sampling fluid (and/or any subsequent sampling fluids) are delivered to a channel on the microfluidic chip.
Advantages of the system of the invention include:
In the description below, the term “microdroplet” or “droplet” incorporates droplets of micro-, nano-, pico-, femto-, and attolitre volume.
The system of the present invention provides for droplet generation, dilution (on-chip or in capillary), merging (on-chip or in capillary), and separation of droplets (on-chip or in capillary). The system comprises:
The system of the present invention comprises a droplet generator, optionally to connected to a microfluidic chip via a section of tubing or a capillary. In the description below, the term ‘tubing’ is used to refer to either tubing or a capillary. The droplet generator comprises an electronically driven microsampling head, comprising an opening (‘microsampling tip’), for aspirating droplets of fluid. The microsampling tip is movable relative to the container, well, reservoir or tank, and optionally movable at least laterally and vertically. The droplet generator opening (microsampling tip) is positioned under a container, well, reservoir or tank, loaded with at least two fluid phases that are immiscible with each other. In one embodiment the droplet generator opening (microsampling tip) is positioned under a container, well, reservoir or tank, that is, the drawing of a sample occurs from below.
When used in a droplet synchronising configuration, the droplet generator comprises at least two electronically driven microsampling tips (openings). The total number of tips corresponds to the number of different fluid sample types being sampled. The tips are driven by an electronic means, such as a motor or an electromagnetic actuator, and are controllable independently of each other. Alternatively, the sample containers are moveable with respect to the microsampling tips.
When the system is configured to enable synchronised sampling and merging of droplets, the number of microsampling tips, affixed to a dedicated section tubing or capillary, corresponds to the number of samples, and each such robotic sampling ‘arm’ is independently controllable.
The tip (opening) may be in the shape of a hollow cylindrical tube, having a first and a second end. The first end of the tip is open and is dipped into a fluid sample contained in a sampling well. A section of tubing is either fixed to the second end of the tip, or the tip may consist of the tubing. The shape of the tip is preferably circular, with either a circular flat or asymmetric opening.
When the tip, or open end of the tubing, is positioned in a fluid phase, the application of pressure (for example, negative and/or positive pressure) causes a volume of the fluid to be aspirated through the tip and into the tubing. Negative pressure is typically applied at a location distant from the tip and positive pressure is typically applied at a location close to the tip.
The system may be configured such that the microsampling tip aspirates fluid samples from a container from below. In one embodiment, the sample-filled container is movable with respect to the microsampling tip, and the microsampling tip is fixed in position. In a second embodiment, the microsampling tip is moveable with respect to the sample-filled container.
By moving the first end of the microsampling tip within a sample-filled container from a first fluid phase into a second fluid phase, and back into the first fluid phase, under application of constant negative pressure, a sample droplet will be aspirated and pulled into the tubing.
The first phase may, for example, be a carrier fluid, and the second phase may be a sample fluid. Repeatedly moving the microsampling tip between the first and second phases creates droplets of sample fluid, interspersed by carrier fluid. The size and spacing of droplets may be determined by the length of time the tip is resident in each phase.
The carrier fluid may, for example, be an oil, immiscible with an aqueous sample fluid, or vice versa. The surface of the sampling component is ideally hydrophobic.
The term ‘sample’ as used herein means a sample of a reagent, compound, substrate, enzyme, cells, or any other component to be analysed.
Reaction volumes are in the pico- to nanolitre range (10−12 to 10−9 L). Droplets may be in the range of 1-100 mL, for example, 10-70 mL, for example, 20-50 nL.
The dead volume (i.e. lost sample) in the present invention is zero, because is sample may be re-used.
In a further aspect, when the system is configured for droplet synchronisation, the droplet generator comprises at least two, for example, at least three, or at least four independently controllable arms, each comprising an electronically driven microsampling tip, and a section of tubing for conducting the droplets to the microfluidic chip. The microsampling tips may all be provided on the same microsampling head, or may each be provided on separate microsampling heads.
In a further embodiment, when the system is configured for droplet synchronisation, the droplet generator comprises at least two, for example, at least three, at least four or at least five, independently controllable arms, corresponding to the number of sample wells or sample types provided.
Preferably, the droplet synchronising system comprises two or three independently controllable arms.
Droplets of each sample type are generated into separate tubing attached to each respective tip. The tubing is connected to a receiving component. The receiving component may, for example, be an extension of tubing, a microfluidic chip, one or more collection vials, a syringe pump, or an analytical instrument. The tubing may also be connected directly to a source of negative pressure (such as a syringe pump) and/or a system outlet.
In one embodiment, the droplet generator will aspirate droplets taken from different samples. Contamination is minimised due to the tip passing through the oil phase, and due to the material of the tip.
In another embodiment, when the system is configured for droplet synchronisation, each tip of the droplet generator is dedicated to use with only one sample, or one type of sample, in order to allow for high speed sampling. For example, a first tip is used for sampling droplets of substrate from a sampling well, or wells, containing a substrate, a second tip is used for sampling droplets of a reagent from a sampling well, or wells, containing a reagent, a third tip is used for sampling droplets of buffer from a sampling well, or wells, containing a buffer.
The samples may be provided in containers carried by a carousel holding a plurality of sample containers, such as Eppendorf tubes. The system of the present invention can also be set up to sample droplets from microtitre plates, such as a 96-well plate, a 384-well plate, or a 1536-well plate. Each sample container, or well, may contain a different sample, for example, each well may contain a different compound from a chemical library. The term ‘container’ will be used to refer to both sample containers and wells in this description.
In use, either a carousel holding the sample containers is rotated (or the well plate is moved) with respect to a microsampling tip, or a microsampling tip can be moved with respect to sample containers.
The microsampling tip of the droplet generator sequentially aspirates droplets from each sample container. The device can be programmed to aspirate one droplet or a plurality of droplets from each sample container. The device may be programmed to aspirate a predetermined different number of droplets from different sample containers.
Droplets from multiple sample containers may be aspirated into the tubing. As an example, one droplet from each of a series of different sample containers may be aspirated, so that a series of droplets, each from a different sample, is aspirated into the tubing. Alternatively, a plurality of droplets (a series) of a first sample A, may be followed by a series of droplets of a second sample B, etc. Each pair of sample droplets is interspersed by a volume of carrier fluid.
A microsampling head and tip, and the tubing (or capillary) attached thereto, which connects the microsampling tip to the microchip, makes up a robotic ‘arm’, which is controlled by a microcontroller. The movement of each arm is independently programmable via the microcontroller which controls retention time in each fluid phase. The whole system has a CPU controlling the pressure and which can feedback to the microcontroller of the robotic arms.
Each section of tubing conducts droplets from the microsampling tip to a microchannel (or capillary) on a microfluidic chip. Separate microchannels on the microfluidic chip converge into one microchannel via convergence points. Alternatively, experiments can be run with droplets which are not merged with another droplet. For example, a reaction may be started just prior to loading the droplets into the system. The reaction progress is then simply monitored in each generated droplet of the reaction mixture.
The present invention enables droplets from a robotic arm to be delivered to a convergence point in a synchronised manner, rather than in a simple flow-dependent sequential manner. The synchronised delivery of 1 to N droplets to a convergence point provides a ‘one-step’ coalescence format based on pressure equilibrium in each of the microchannels.
This synchronised arrival time of droplets from the microchannels at a convergence point enables two or more droplets to merge into one ‘mixed droplet’. A reaction may be initiated in each mixed droplet upon merging, by the sample and the reagent coming into contact with each other. This can also occur directly in capillary. Each synchronised arrival of one droplet from each of the microchannels at the convergence point leads to the creation of one mixed droplet. Sequential mixed droplets in the series are completely separated from each other by a volume of the oil (carrier fluid).
This synchronisation allows droplets to arrive at a convergence point at a predetermined time. Thus, not only can two or more droplets be controlled to arrive at the convergence point at a substantially identical time, but the time of arrival of each droplet may also be accurately controlled, allowing a first and second droplet to arrive at a convergence point a predetermined length of time apart. Optionally three or more droplets can be merged by arriving at a convergence point at the same time.
Each (optionally mixed) droplet proceeds along a channel on the microfluidic chip to a receiving component where the progress of a reaction can be monitored. Alternatively, each optionally mixed droplet is monitored in capillary. Alternatively, the droplet may proceed along the channel to a receiving component for storage. The receiving component may be a length of tubing. This tubing may be connected at one end to the outlet of the system or to a channel on a microfluidic chip, or it may be a microchannel or microstructure on a microfluidic chip. Droplets (optionally mixed) may alternatively be deposited on a surface, e.g. a MALDI plate, a bead plate for emulsion PCR, or be sprayed, e.g. in electrospray ionisation.
In the prior art, sequential arrival of droplets in a microchannel suffers from the necessity of drawing sample and reagent alternately, and thus necessitates the inclusion of a wash step to minimise or avoid cross-contamination of the different samples. The sequential arrival systems of the prior art also suffer from time delay in between the droplets, which limits the throughput of the system.
With the present invention, high sample throughput is possible. The invention allows for creating a sequence of identical droplets (e.g. reagent or sample) in one capillary for subsequent merging with a series of non-identical droplets (e.g. sample or reagent) and, also for merging these droplets with a series of droplets of a further component, such as a further reagent, or a buffer. These droplets may be identical or non-identical, and may be of the same or a different volume to each other.
Independent control over each of the robotic arms of the microfluidic system is possible, to control frequency of sampling, droplet volume, droplet spacing, number of capillaries being used, speed of droplet flow etc.
Merging of droplets can be pre-programmed for timed reactions to occur. For example, a first droplet (A) and second droplet (B) can be merged first, before the resulting mixed droplet (A+B) is merged with a third droplet (C). Alternatively, droplets A, B and C can be controlled to arrive at a convergence point at precisely pre-determined time-spacings (in-capillary or on-chip) to merge droplets A+B+C together at the same time. By controlling the movement of the droplets in this precise way, no droplet of sample will miss receiving a volume of reagent (and/or buffer), thereby achieving reliability of experimental results.
Merging sequences of droplets can be programmed for timed reactions. Further droplet merging may be carried out. Size, material and design of the tubing or microfluidic channels are important to achieving this function.
Droplet generation can be achieved from below as shown in
In
In a further alternative embodiment, the base of the sampling chamber shown in
In one aspect, fluid (b) is an oil and fluid (a) is a sample. In a further embodiment, if the container is open above the sample, sample fluid (a) may be covered with an oil layer, with an oil with a density lower than the sample, to prevent evaporation of the sample.
The provision of an oil layer on top of and/or beneath the sample fluid avoids the need for a washing fluid. Tests carried out by the Applicant have shown that there is no cross-contamination between droplets sampled by the device of the present invention.
The systems described in
Approaches for droplet merging use either passive, active or passive/active mechanisms. Passive droplet trapping relies on the use of geometrical constraints to bring two or more droplets into contact. These include inter alia channel enlargement, use of pillar arrays, use of hydrophilic patches, surface tension, viscosity, size can also be used to achieve merging. Active merging mechanisms include inter alia electric fields (electrocoalescence), magnetic fields, acoustic waves, pneumatic valves. Hybrid droplet merging, combining pillars and electric fields, has been reported in the literature.
Referring to
Merging may be carried out by a variety of means. For example, merging may be assisted by the provision of a merging chamber (40), which retains a first droplet between a series of parallel pillars (41), the pillars allowing the carrier fluid to flow around (42) the pillars, such that a second droplet, and optionally a third droplet, merge into a single droplet (35), which, due to surface tension and the pressure applied to the system, then flows out of the merging chamber. The surface properties of the pillars, tubing and walls of the merging chamber, as well as the spacing of the pillars and size of the merging chamber, are determinative for this process. Other droplet merging mechanisms can also be implemented, such as by channel widening to slow down the progress of large droplets; or other mechanical, electrical or optical droplet trapping approaches.
Importantly, the merging of these droplets is controlled, due to the controlled, synchronised delivery of the droplets from the robotic arms to the channels of the microfluidic chip. The invention allows for, and guarantees that, each droplet in a series of droplets will be merged with the intended droplet from a second series of droplets, generated by a separate robotic arm. The controlled merging achieved by the system of the present invention ensures that no droplet fails to receive its intended merger droplet. In one embodiment, one droplet may be, e.g., a substrate, a second droplet may contain a first sample, e.g., a buffer solution, and a third droplet may contain a second sample, e.g., a reagent. As an example, a first sample well (3) contains a substrate, a second sample well (3) contains a reagent, and a third sample well (3) contains a buffer.
To induce droplet flow through the device, negative and/or positive pressure is applied to the system.
Negative pressure is typically applied at the end of the system, or microfluidic chip, distant from the droplet generator, such that the droplets are pulled through the entire system. A suitable negative pressure is in the range of 0-100 Pa, or the application of 0.001-0.01 atm of negative pressure (beyond ambient pressure).
Additionally, or alternatively, positive pressure may be applied to the samples to drive the sample droplets through the system. This positive pressure may be the same or different for each of the robotic arms, and is controllable independently in each arm. Positive pressure is typically applied at the end of the system proximate to the droplet generator, such that the droplets are pushed through the system. A suitable positive pressure is in the range of 0-100 Pa, or the application of 0.001-0.01 atm of positive pressure (beyond to ambient pressure).
In a further embodiment, the system may comprise further points in the system—besides at one or both of the ends of the system distant from or proximate to the droplet generator or synchroniser—where pressure may be applied. In such an embodiment it is essential that the system can be is selectively and reversibly sealed between different parts of the apparatus corresponding to different pressure phases. Additional application of positive or negative pressure may assist droplet flow.
The system should be sealed to maintain applied pressure and this can be achieved by means known to the skilled person. Air-tight syringes and air-tight tubing to pump connectors may be used.
Another advantage of the system of the present invention is that no valves are necessary to move sample droplets through the system in either the tubing or the microfluidic chip channels. This leads not only to a much simpler design, but also to a faster operational capability than a system including valves. In contrast, the prior art uses valves to control the flow of droplets. This can lead to contamination between droplets.
The devices of the system of the present invention are fabricated in a material that has a particular wettability with respect to the continuous phase. That is, the oil phase should wet the channel surfaces and the aqueous droplets should not wet the channel surfaces. Suitable materials include glass, plastics material, and polymers, including polymeric organosilicon compounds.
The devices may, for example, be fabricated from polydimethylsiloxane (PDMS) using known soft lithography methods. The interfacial tension between PDMS and water is approximately 40 mN/m at room temperature. The interfacial tension between PDMS and oil is approximately 1-39 mN/m at room temperature.
The interior wall of the tubing is coated with a material to lend the tubing suitable surface properties. A suitable example for many applications is polytetrafluoroethylene (PTFE; Teflon). Alternatively, the tubing can be made entirely of PTFE or one or more similar materials, such as fluorinated ethylene propylene (FEP), perfluoroalkoxy (PFA). For applications such as single cell studies, the tubing may be coated with a suitable material, such as: gelatine, extra-cellular matrix, collagen, agarose, alginate, poly-L-lysine, phosphatidyl-choline, growth factors, glutamate, BSA, Aquapel. Important properties are, for example, hydrophobicity, binding capacity, biological interactions.
The tubing has an inner diameter of between 1 μm to 1 cm, for example, between 20 μm to 1 mm, between 20 μm to 500 μm, between 200 μm to 400 μm for example, approximately 300 μm. The tubing has a length of between 1 cm and 1000 cm, for example between 10 cm and 100 cm. This tubing may subsequently be connected to a further device for off-line separation.
The minimum size of droplet for the present invention is such that the droplet completely separates two sequential plugs of carrier fluid, i.e. the droplet must have contact with at least a circumference of the inner diameter of the tube. Minimum droplet size for a particular size of tubing can be determined by the following mathematical formula:
wherein V is the volume of the droplet, and r is the inner radius of the tubing.
For the present invention, the internal radius of tubing is preferably in the range of 1 μm to 1 cm. For tubing having an inner diameter of 100 μm, the to minimum droplet volume is approximately 0.52 nL.
For a droplet that does not have a perfectly spherical shape, the droplet volume approximates to that of a cylinder with hemispherical ends:
V=(π·r2·L)+(4·T/3·r3)
wherein L is the length of the cylinder and r is the inner radius of the tubing.
The carrier fluid may be an oil. Important surface properties of the oil include density, dynamic viscosity, water solubility, thermal conductivity, and boiling point. Examples of suitable oils include, but are not limited to, fluorinated oils, non-fluorinated oils, mineral oils, plant oils, vegetable oils, comestible oils. For example: fluorinated oils such as fc40 or fc328, mineral oil, oleic acid, embryo-tested mineral oil, light mineral oil, heavy mineral oil, PCR mineral oil, AS4 silicone oil, AS 100 silicone oil, AR20 silicone oil, AR 200 silicone oil, AR 1000 silicone oil, AP 100 silicone oil, AP 1000 silicone oil, AP 150 silicone oil, AP 200 silicone oil, CR 200 Silicone oil, DC 200 silicone oil, DC702 silicone oil, DC 710 silicone oil, octanol, decanol, acetophenone, perfluoro-oils perfluorononane, perfluorodecane, perfluorodimethylcylcohexane, perfluoro-1-butanesulfonyl fluoride, perfluoro-1-octanesulfonyl fluoride, perfluoro-1-octanesulfonyl fluoride, nonafluoro-1-butanesulfonyl chloride, nonafluoro-tert-butyl alcohol, perfluorodecanol, perfluorohexane, perfluorooctanol, perfluorodecene, perfluorohexene, perfluorooctene, fuel oil, halocarbon oil 28, halocarbon oil 700, hydrocarbon oil, glycerol, 3M Fluoriner fluids (FC-40, FC-43, FC-70, FC-72, FC-77, FC-84. FC-87, FC-3283), soybean oil, castor oil, coconut oil, cedar oil, clove bud oil, fir oil, linseed oil, safflower oil, sunflower oil, almond seed oil, anise oil, dove oil, cottonseed oil, corn oil, croton oil, olive oil, palm oil, peanut oil, bay oil, borage oil, bergamot oil, cod liver oil, macadamia nut oil, camada oil, chamomile oil, citronella oil, eucalyptus oil, fennel oil, lavender oil, lemon oil, nutmeg oil orange oil, petitgrain oil, rose oil, tarragon oil, tung oil, basil oil, birch oil, black pepper oil, birch tar oil, carrot seed oil, cardamom oil, cassia oil, sage oil, cognac oil, copaiba balsam oil, cypress oil, eucalyptus oil, dillweed oil, grape fruit oil, ginger oil, juniper oil, lavender oil, lovage oil, majoram oil, mandarin oil, myrrh oil, neroli oil, olibanum oil, onion oil, paraffin oil, origanum oil, parsley oil, peppermint oil, pimenta leaf oil, sage oil, rosemary oil, rose oil, sandalwood oil, sassafras oil, spearmint oil, thyme oil, transformer oil, verbena oil, and rapeseed oil.
Droplets may be stored as dispersed in the oil phase in the tubing or microfluidic droplet connector, for later use in further analysis or experimental techniques.
The present invention finds application in high-throughput screening, monitoring and analysing reaction kinetics, diagnostics, drug screening (kcat, kI, kM, IC50, EC50), single cell screening and personalised medicine, such as biomarker screening on small tissue samples, cancer screening, patient profiling etc.
There are many advantages associated with the system of the present invention, in terms of sample volume needed, number of assays that can be run per day, and cost. In conventional microfluidic systems, at least 100 μl of each sample is required, and only 1-10 samples can be tested per day. The repeat rate is high at 108. In High-Throughput Screening (HTS), a single assay requires 100 nL-50 μL of sample. Approximately 104 experiments (i.e. individual data points) can be run per day. However, the cost of HTS is very high, at around $10-100 million for set up and $10,000-$100,000 in maintenance and running costs per year.
In comparison, the system of the present invention has the potential to screen hundreds of samples (e.g. compounds to be tested) per day, and can produce 104 more assays per day than conventional microfluidic systems. Further, the system of the invention can produce 104 samples (e.g. of compounds to is be tested) per day, and one sample can be used for 108 assays. With the system of the present invention, 100 assays can be performed per sample (e.g. a drug) per day. Accordingly, thousands of assays can be run per day at a rate of 1 to 100 Hz. The system of the present invention generates approximately 10 droplets per second (10 Hz) per microsampling tip, but can potentially reach much higher frequencies of droplet generation if on-chip splitting of droplet is combined, (e.g. up to 1 kHz).
Another major advantage of the present invention is that only a very small amount of starting sample—about 15-40 μl—is needed. In the sampling system of the present invention, a droplet may typically be around 1 nL in volume. This is sufficient for one repeat of one experimental test. Accordingly, the present sampling system allows for 15.000-40.000 droplets to be generated, and 15.000-40.000 tests run from one sample. This compares highly favourably to current methods of High Throughput Screening (HTS) or conventional microfluidics. The present invention requires 103-106 times less sample volume than HTS.
The sample containers may comprise at least one layer of sample fluid and one layer of carrier fluid. For example, the sample containers may comprise one, two or three layers of sample fluid, in addition to carrier fluid. The layers remain separate on account of different densities of the different fluids. In one embodiment, a layer of carrier fluid may separate two layers of different sample fluids, in a sample container.
The system of the present invention incorporates a very high degree of flexibility in droplet manipulation options. Various illustrative examples are discussed below. This discussion is not intended to be limiting on the scope of the claims.
Droplet size and carrier fluid volume can be determined by the residence time of the tip of the microsampling head in the sample solution. The timing is predetermined by programming the electronic means controlling the movement of the microsampling tip and/or of the sample container(s). The minimum droplet size for the present invention is such that the droplet completely separates two sequential plugs of carrier fluid, i.e. the droplet must have contact with at least the inner circumference of the tubing or microchannel.
In any given experiment, droplet size is controllable. For example, all of the droplets may be of substantially the same size, e.g. all droplets generated in a particular experiment will be of substantially the same volume. Alternatively, all droplets of a particular type will be of substantially the same volume, and droplets of different types may have a different volume. Alternatively, all droplets of a particular type may have a different volume.
An example of this is droplets containing an enzyme each being of a first size, and droplets containing a substrate each being of a second size. Droplets of the same type may also be of different sizes, for example, when different amounts of a component are needed, such as a greater amount of a substrate, or different amounts of buffer, in order to create different levels of concentration in a droplet.
By the same means, the volume of the intervening carrier fluid can also be precisely controlled. This precision in droplet size and droplet separation achieved by the sampling capability of the droplet generator allows for precise control on merging two or more droplets together.
Merging may be carried out by various methods. Directly in-capillary or in a channel on the microchip, where merging of two or more sequential droplets can be induced by providing a wider section of the channel, a pillar arrangement, or by other methods of droplet trapping. When increasing the internal diameter of the tubing or capillary for an appropriate distance, two or more sequential droplets merge, as the droplets no longer fill an entire cross section of the tube due to hydrophilic attraction. The number of droplets that will merge is dependent on the length of the wider section of the tubing and the separation distance between sequential droplets. The resulting merged droplet will be pulled into the narrower tubing section by the negative pressure applied to the system.
Another merging technique allows droplets to merge in channels on the microchip, where channel junctions exist, as shown in
The present invention allows for precise control of the arrival time of the droplets at merging sites. The present invention ensures that in two separate series of droplets to be merged, each droplet in the first series will arrive at the merging site at the same time as each ‘corresponding’ droplet from the second series. Due to the precise control over droplet size and droplet to spacing achieved by the robotic arms, the present invention allows for precise timing control. This ensures that each droplet from a first series will be merged with a droplet from the second series. Merged droplets will continue to flow through the system.
Where pairs of droplets to be merged contain species that will react with each other, the precise merging also ensures that each droplet pair will be correctly merged as intended, and also allows for the precise determination of the start time of a reaction. This precision is crucial for accurately determining the extent and rate of a reaction.
Various analytical data can be gathered from carrying out such studies. Another advantage of the invention is that thanks to the small sample volumes and lower cost of running the experiments, it is easily possible to obtain vast is amounts of very accurate data. Very small changes to the concentration gradient of a substrate can easily be made within a droplet series. This guarantees that the resulting data is extremely accurate and that no data points are missed.
4. Flexibility within One Experimental Run
The system of the present invention incorporates a high degree of flexibility. This is possible due to the highly precise way in which the droplet sampling and synchronised merging can be controlled. The microsampling tip may sample a single droplet from each compound sample to yield a series of different droplets (e.g.
If droplets of different sizes are aspirated by the microsampling tip, the on-chip or in-capillary dilution and/or on-chip or in-capillary merging capabilities enable all the droplets in the series to be brought to the same size. This is, for example, achieved by merging different volumes of buffer solution with the sample droplets. The present invention enables precise control, allowing the droplets of the buffer solution aspirated by the microsampling tip to be of the exact volume needed to bring each droplet in the sample series to the same size. The system also allows for no buffer to be added to any sample droplets that do not require any buffer. The device is accurately controllable to ensure that the merging of sample and buffer droplets occurs correctly.
The microsampling tip can also aspirate a plurality of droplets from each sample well. An example use of this would be in conjunction with a dilution study, as described above. For example, a number of droplets of sample A can be aspirated. These may be of an identical size, and hence of an identical concentration. A varying volume of buffer solution can then be added to each droplet (e.g. increasing or decreasing along the series) in order to adjust the size (and relative concentration) of each droplet. Alternatively, the sample droplet concentration can be increasing or decreasing along a series, by aspirating droplets of increasing or decreasing size. An appropriate volume of buffer solution can be added to each droplet in order to adjust the droplet size.
Droplets may be merged with a droplet of a reagent that will react with the species in the sample droplet. This can be carried out in various ways. For example, a series of droplets each containing a different compound from a chemical library can be merged with a droplet containing a specific reagent.
An example of such an application would be in a chemical reaction, such as an enzyme reaction.
A series of identical sample droplets, of an identical concentration, can be merged with a series of droplets containing a differing amount of reagent, for example, the droplets may contain an increasing or decreasing reagent concentration. This enables the lowest feasible reagent concentration to be determined, and also enables the determination of the effect of concentration on reaction rate.
Similarly, a series of identical sample droplets of different concentrations may be merged with a series of reagent droplets each having an identical concentration.
In another embodiment, a series of identical sample droplets of different concentrations may be merged with a series of reagent droplets containing a differing amount of reagent. Each such experiment produces valuable and is accurate data.
A further advantage of the present invention is that droplets do not need to be labelled, because they remain in the same sequence in the tubing or microchannels. The present invention thereby removes any need for incorporating markers or labels in the samples. This is particularly useful in monitoring reaction rates and biological reactions.
Using the system of the present invention, droplets containing different reactants can be merged. Upon merging two droplets, each containing a different reagent, a reaction between the reactants can be initiated. The progress of this reaction can be monitored in real time. For example, if a droplet comprising a first reagent is merged with a droplet comprising a second reagent, a reaction between the first and second reagent will take place. This reaction can be monitored using techniques such as fluorescence imaging, laser detection methods, Raman scattering or label-free detection such as absorbance, changes in refractive index or bioluminescence.
Series of droplets can also be repeatedly run forwards and backwards across a laser or fluorescence beam in order to analyse the time course of the reaction. Data from each individual droplet can then be gathered.
It is also possible to selectively consider only droplets of interest. For example, it is possible to study only droplets containing a first and second reagent at specific concentration levels of interest. This study gives data on the relationship of the reaction rate with the concentrations of each reagent.
7. Continuous Screening (on-Chip or in Capillary)
Sequences of droplets as micro-reactors are analysed for a readout (fluorescence, absorbance). The readout measurement can be taken continuously over a time-course or at specific time points either in continuous-flow, or statically with droplet trapping or storage. Detection of the reaction can be done either on chip or in capillary. Because the droplets are analysed in sequence, multiple readouts can be assigned to each droplet giving real-time data.
In single cell screening, such as biopsies, or tissue samples, and screening clinical trial candidates, one cell is contained in one droplet. This allows for the screening and monitoring of a single cell. The data obtained from these single cell experiments is highly specific and therefore extremely valuable. The isolation of single cells into droplet compartments yields more accurate statistical data. Current experimental techniques allow for averaging of data over 3000 cells. The system of the present invention allows for the screening of much larger numbers of cells (up to 106 to 108 cells). In general, detection of cell assays (e.g. signalling, surface binding) through fluorescence or absorbance measurements can be used for toxicity prediction, stem cell studies, diagnostic applications (e.g. Cancer screening and personalised medicine, for diseases, including, but not limited to Alzheimer's Disease, Malaria).
The present invention allows for the development of assays for screening a multitude of samples or analytes across a variety of applications and disciplines. These encompass:
Analysis of droplets can be carried out by various methods. Appropriate detection techniques include high-speed imaging, Laser detection, absorbance and luminescence (fluorescence, phosphorescence, chemi- and bio-luminescence).
It is also possible to control the droplets to be moved forwards and backwards over a point of detection, such as a laser, in order to obtain data for each droplet over a time course. This can be achieved by adjusting the pressure in the system. This is illustrated in
Further data for the examples described below are shown in
This example relates to the reaction of enzyme β-glucosidase and different concentrations of substrate RDGlu (Resorufin-D-Glucopyranoside) in buffer PBS (100 mM, pH 7.4). The reaction product is resorufin, a fluorescent dye. The system configuration is shown in
As a first step, β-glucosidase is applied to a cover-slip, with the lowest and highest RDGlu concentrations to be used. This enables the determination of the fastest and slowest time taken to reach saturation. Then, samples of β-glucosidase, and samples with different concentrations of RDGlu are made up immediately prior to loading the samples into the droplet generator. Droplet sequences of RDGlu with different concentrations are generated. The droplet synchroniser generates droplets of the enzyme and buffer. On a merging chip, 10 to 50 repeats of each reaction of a different RDGlu concentration and β-glucosidase and buffer are merged. Droplets flow off the microfluidic chip into a section of tubing, where analysis and reaction monitoring is carried out by laser detection methods. Droplets reach the laser approximately 30-120 seconds after droplet generation. Droplets are run back and forth over the laser to yield time course data.
The reaction of Example 1 is repeated, this time generating droplets of different concentrations of an inhibitor and food dye in the droplet generator, and generating droplets of buffer, enzyme, and different concentrations of RDGlu in the droplet synchroniser. On a merging chip, 10 to 50 repeats of each reaction are merged (
The reaction of Example 2 is repeated, this time generating droplets of an inhibitor from a stock solution, using buffer to dilute the concentration, and food dye in the droplet generator, and generating droplets of buffer, enzyme, and RDGlu from a stock solution in the droplet synchroniser. On a merging chip, 10 to 50 repeats of each reaction are merged (
The reaction of Example 1 is repeated, this time generating droplets of RDGlu from a stock solution, buffer and food dye in the droplet generator, and generating droplets of buffer, enzyme, and substrate in the droplet synchroniser. Fluorescence imaging is used to determine the extent and rate of the reaction in the droplets. On a merging chip, 10 to 50 repeats of each reaction are merged (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1211342.9 | Jun 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/051668 | 6/25/2013 | WO | 00 |
