GB2459085A

GB2459085A - Air-segmented micro-mixing system for chemical and biological applications

Info

Publication number: GB2459085A
Application number: GB0806165A
Authority: GB
Inventors: Patrick Douglas Shaw Stewart
Original assignee: Shaw Stewart P D
Current assignee: Shaw Stewart P D
Priority date: 2008-04-07
Filing date: 2008-04-07
Publication date: 2009-10-14
Also published as: GB0806165D0

Abstract

A versatile air-segmented micro-mixing system with chemical and biological applications The present invention provides microfluidic technology for mixing discrete volumes of chemical and biological solutions and suspensions in an accurate, versatile and predetermined manner. There are a plurality of liquid channels 13 feeding into a mixing conduit 10. Various chemical solutions may be introduced into the mixing conduit 10. Slugs of liquid may be kept separate and moved along the conduit by introducing gas or air bubbles from gas channels 14. The slugs may be detected by a detector 15 and dispensed by a dispensing tip 16.

Description

Versatile air-segmented micro-mixing system with chemical and biological applications

TECHNICAL FIELD

This invention relates to microfluidic systems, and in particular to mixing discrete aliquots of chemical and biological solutions and suspensions in an accurate, versatile and predetermined manner. The volumes to be mixed range from less than a femtolitre to a few milliliters.

BACKGROUND OF THE INVENTION

Microfluidic systems have the advantages of using very small samples, consuming small amounts of samples and reagents, and compact size.

Many applications have been suggested for microfluidic systems. Applications and suggested applications include protein crystallization, DNA analysis, DNA synthesis, genomics, cell based systems, clinical diagnostics, bio-defense sensors, high throughput screening, pharmaceutical lead optimization, high content screening, combinatorial chemistry and genetic fingerprinting.

In 1957 Leonard Skeggs invented a special flow analysis technique named "continuous flow analysis" (CFA) that was commercialized by Technicon Corporation. This method is disclosed by Skeggs in US patent number 2797149, issued in 1959. CFA generally used air to segment the flow of reagents and samples. The method has the disadvantage that each sample is divided into several segments, where the contents of each segment is not individually controllable. Another disadvantage is that the method tends to consume large amounts of reagents.

A similar approach was developed by the inventor of the present invention in W08402000 and GB patent 2097692B in 1982. These documents described a "Chemical Droplet Reactor". Here samples, reagents etc. are dispensed into oil in a conduit system as individual droplets. Liquids can be added to samples, which can be heated, incubated, analyzed etc. Each droplet is equivalent to a test-tube and it is dealt with individually. Also, GB2097692B teaches miniaturization and the construction of conduits by forming depressions in the surface of one or more sheets and bringing these sheets into face-to-face contact.

Improved methods for dispensing, priming and calibrating a microfluidic device, and also novel hardware were later disclosed by the present inventor in patent application GB0704786.3.

The approach of GB2097692B and other more recent inventions that use oil as a carrier liquid suffer from the disadvantage that if any samples or reagents come into contact with the walls of conduits it becomes difficult to clean the conduits. The current invention seeks to combine the best aspects of CFA with the best aspects of the Chemical Droplet Reactor. It seeks to provide a versatile system for combining liquids without waste that is also easy to flush and clean.

BRIEF SUMMARY OF THE INVENTION

The present invention aims to satisfy the long-felt need for microfluidic devices that are easily adaptable to novel research and testing applications. The invention is able to generate multiple discrete liquid aliquots, where each aliquot contains a mixture that is individually controllable and can be unique. For example, it can set up combinatorial experiments using dozens of ingredients, where every mixture is determined using a computer program for experimental design. These aliquots take the form of discrete volumes "bracketed" by gas bubbles that are situated upstream and downstream of each aliquot.

One aspect of the invention is a microfluidic apparatus with reservoirs filled with chemical or biological solutions, emulsions or suspensions (hereinafter referred to as "chemical solutions"). These reservoirs are in communication with a conduit where mixing of the chemical solutions takes place. This mixing conduit is initially at least partially filled with a system liquid. The system liquid is at least partially miscible with at least some of the chemical solutions. The system liquid could be water, buffer or a solvent such as methanol, ethanol, acetone, DMSO etc. It could also contain salts, polymers, detergents, lipids, and many other solutes. Oil-based system liquids such as paraffin, silicone or fluorinated organic liquids can be used for oily samples and reagents.

In addition to reservoirs for chemical solutions and a mixing conduit, the apparatus comprises a conduit for introducing gas into the mixing conduit.

It may also possess a detector that can detect the presence of liquids or gas bubbles, and a dispensing tip.

A further aspect of the invention is a method of using the detector to calibrate the system with regard to accurately dispensing the solutions into the mixing conduit.

Another aspect of the invention is a dispensing tip that possesses a piece of hardware referred to hereinafter as a "nosebag" that allows fluids to pass out of the tip and to be taken to a waste receptacle.

Yet another aspect of the invention is a method of using the dispensing tip and the nosebag to dispense liquid aliquots to a sample receptacle while transferring the fluid that is downstream and upstream of the each liquid aliquot to waste.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic view of a generalized embodiment of the invention that has liquid channels for five chemical solutions, a gas channel, a liquid channel for introducing system liquid, a detector, a dispensing tip, and a device for collecting fluids from the dispensing tip called a nosebag.

Figures 2a to 2g show a method of mixing chemical liquids. Three chemical liquids are selected from the five chemical liquids that are available.

Figures 3a to 3d show a method of calibrating the system using a time-and-pressure approach.

Figure 4a to 4e show a method of dispensing a liquid aliquot to a sample receptacle while transferring the fluid that is downstream and upstream of the each liquid aliquot to waste.

Figure 5 shows a design that allows the inlets of liquid channels to be spaced relatively far apart, while still allowing the openings of liquid channels into the mixing conduit to be close together.

Figure 6 shows that the liquid channels can posses constrictions (61) at the openings (9) of reservoirs (13) where the reservoirs join the mixing conduit (10).

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, with reference to the accompanying drawings.

In the accompanying drawings, like features are denoted by like numerals.

Figure 1 is a schematic view of a generalized embodiment of the invention that has liquid channels (13) for five chemical solutions, a gas channel (14), a liquid channel for introducing system liquid (11), a detector (15), a dispensing tip (16), and a device for collecting fluids from the dispensing called a nosebag (17 and 18). Each liquid channel comprises a source of pressure (not shown) and a valve (12) or other device for moving fluids. Pumps, syringes, pistons etc. can be used instead of valves or as well as valves.

The gas channel (14) is shown as having a smaller cross-section than the other channels in order to provide greater resistance to flow, since gasses generally have much lower viscosities than liquids. A detector (15) that is adjacent to the mixing conduit is able to distinguish between liquids and gasses in the mixing conduit. The nosebag has an upper and a lower position. When the nosebag is in the upper position (17), the dispensing tip (16) can be used to dispense liquid aliquots to a suitable receptacle or receptacles. When the nosebag is in the lower position (18), all fluids that pass out of the dispensing tip are withdrawn into the nosebag, and they pass out of a side-arm to waste (19).

Figures 2a to 2g show a method of mixing chemical liquids. Three chemical liquids are selected from the five chemical liquids that are available. At the start of the sequence, the mixing conduit (10) is full of system liquid (28) or of another fluid. Air or gas is introduced into the mixing conduit from the gas channel, advantageously forming a gas bubble as shown figure 2a. (In some cases it may now be necessary to move the gas bubble by introducing system liquid from the system liquid channel. This step is not shown because it is not necessary in the example shown.) In figure 2b, solution is introduced into the mixing conduit from a first liquid channel. The solution introduced (22) divides the gas bubble into an upstream bubble (23) and a downstream bubble (24). In figure 2c system liquid is added from the system liquid channel so as to move the bubbles and the solution introduced further down the mixing conduit. In figure 2d a second chemical solution (25) is introduced into the mixing conduit, where it mixes with the first solution (22). After this a third chemical solution (26) is introduced as shown in figure 2e. In figure 2f, more system liquid is introduced to move the bubbles and the solutions (22+25+26) away from the liquid channels. After this, the mixing conduit can be cleaned as shown in figure 2g. System liquid and gas are introduced into the mixing conduit. The gas is helpful because it removes fluid that might otherwise remain in contact with the walls of the mixing conduit. The gas can be introduced simultaneously with the system liquid, or the two fluids can alternate. The mixing conduit could also be cleaned in a similar way by introducing a cleaning fluid. (Suitable ingredients for cleaning fluids include alkalis, acids, detergents, enzymes, cleaning agents, solvents etc,) Figures 3a to 3d show a method of calibrating the system using a time-and-pressure approach. The time-and-pressure approach is one of several possible methods of using the system. Other methods will be discussed below. In figure 3a, a gas bubble (21) is introduced into the mixing conduit (10). In figure 3b, solution (31) from a first liquid channel is introduced into the mixing conduit (10), forming a liquid aliquot and dividing the gas bubble into an upstream bubble (23) and a downstream bubble (24). System liquid is now introduced to the mixing conduit until a detector (15) detects the transition from the downstream bubble (24) to the liquid aliquot as shown in figure 3c. In figure 3d, still more system liquid is introduced until the detector (15) detects the transition from the liquid aliquot to the upstream bubble (23).

The volume of system liquid that is introduced to the mixing conduit between the situation shown in 3c and 3d is approximately equal to the volume of the chemical solution that was introduced at 3b. If the volume of system liquid that is introduced is not directly known, the time interval between 3c and 3d can in some cases be used to calibrate the system.

In one approach, the system liquid is itself introduced by a time-and-pressure method. For example, a reservoir of system liquid could be maintained at constant pressure and a valve could be opened to allow the system liquid to flow. The time taken for the air-bubbles to flow past the detector is now recorded.

Note however that it may be difficult to estimate the volume of chemical solution dispensed by such an approach. For example, if the upstream bubble passes the detector long after the downstream bubble, this may indicate that the solution dispensed has low viscosity because a large volume has passed into the mixing conduit. However, it could indicate that the solution dispensed has a high viscosity because it takes a long time to flow past the detector. Several approaches are possible to resolve this difficulty. For example, the system liquid channel could be made with a higher resistance to flow than the solution channel. Therefore the viscous resistance in the mixing conduit becomes unimportant. Or the mixing conduit could be made with a lower resistance to flow (i.e. larger diameter) than either the system liquid channel or the solution channel. A third approach is to move the system liquid with a positive displacement device. Examples of positive displacement devices are certain gear pumps, syringes or pistons, where the volume moved is independent of the back-pressure.

In a second approach, the system liquid is moved by a positive displacement device such as a syringe, a piston or a positive displacement pump such as a gear pump, so that the volume of system liquid introduced is well-known. In this method the chemical solutions are still moved using a time-and-pressure approach.

Advantageously each liquid channel can be tested several times with different volumes being dispensed, and the results can be combined.

A simple approach would assume that the volume dispensed is proportional to the time that the valve is open. A more complex approach would use a look-up table to relate the volume dispensed to the time that the valve is open.

Note that all of these techniques take into account both the geometry of the device and the properties (particularly viscosity) of the chemical solutions.

Yet another approach is to use positive displacement devices to move all of the liquids. Such a device is likely to give greater accuracy, but may be more difficult to construct. Here there is no need to calibrate the system.

Figures 4a to 4e show a method of dispensing a liquid aliquot to a sample receptacle while transferring the fluid that is downstream and upstream of the each liquid aliquot to waste. In figure 4a a liquid aliquot (41) to be dispensed into a receptacle is situated in a conduit and defined by an upstream bubble (23) and a downstream bubble (24). The nosebag is in the lower position (18). System liquid moves into the conduit, moving the liquid aliquot and the bubbles towards a dispensing tip (16), and moving liquid out of the dispensing tip and into the cavity of the nosebag. Suction is applied to the side-arm (42) of the nosebag, so that the liquid in the cavity is carried out of the side-arm to waste.

This movement continues until the detector (15) detects the end of the downstream bubble (24) (i.e. the beginning of the liquid aliquot) as shown in figure 4b. Now the movement continues again until it is reckoned that at least part of the downstream bubble has passed out of the dispensing tip (16), as shown in figure 4c. (At some point before or after 4c the detector detects the lagging end of the liquid aliquot.

This event is not shown in the figures.) Now the nosebag is moved from the lower position (18) to the upper position (17), and the tip is moved into a receptacle (43) such as the well of a 96-well plate as shown in figure 4d.

In figure 4e the aliquot is moved into the receptacle (43) to form a drop (44). At the same time, the dispensing tip may move out of the receptacle as shown -this improves the accuracy and regularity of dispensing. When the upstream bubble reaches the tip, liquid movements are stopped. The dispensing tip is then moved out of the receptacle.

Note that the movements described are relative. For example, instead of moving the nosebag up and keeping the dispensing tip still, it would be equally effective to keep the nosebag still and to move the dispensing tip down. Another example is that moving the tip down into a (stationary) receptacle would be equivalent to moving the receptacle up to a (stationary) dispensing tip.

In the approach described, the position of bubbles is determined by the detector. However this is not necessary. Instead, the position of bubbles can be estimated by dead reckoning.

Figure 5 shows an example of a design that allows the inlets of liquid channels to be spaced relatively far apart, while still allowing the openings of liquid channels into the mixing conduit to be close together.

Ten liquid channels (53) are connected to a mixing conduit (54) as close together as possible. Another channel (51) is provided for introducing gas to the mixing conduit (54). The mixing conduit has an inlet (52) for introducing system liquid, a detector (56) and an outlet (57). The inlets of the ten liquid channels and the gas channel are placed along a line that is at right-angles to the mixing conduit. This configuration and other similar configurations allow the connections to the mixing conduit to be close together, while the inlets to the channels are relatively far apart. Again, the gas conduit is longer an nanower than the other conduits.

Figure 6 shows that the liquid channels (13) can posses constrictions (61) at the openings (9) of reservoirs where the reservoirs join the mixing conduit (10). These constrictions can clearly reduce the amount of material that can accidentally come out of the opening by laminar flow or by diffusion etc.

Claims

CLAIMSWhat I claim is: 1. A microfluidic system for combining chemical solutions comprising an apparatus and a method, where the apparatus comprises: (a) a mixing conduit having a distal and a proximal end, (b) an inlet at the distal end, (c) an outlet at the proximal end, (d) a plurality of liquid channels, where each liquid channel comprises an opening into said mixing conduit, a side conduit in fluid communication with said mixing conduit via said opening, and a device for moving fluids in said side conduit, and (e) a gas channel for introducing air or gas into said mixing conduit; and the method comprises the steps of: (a) loading chosen chemical solutions into said side conduits, (b) filling or partially filling the mixing conduit with a system liquid that is at least partially miscible with the chemical solutions that are to be mixed by the system, (c) moving a bubble of air or gas from said gas channel into the mixing conduit, (d) if required, moving said system liquid until said bubble is adjacent to the opening associated with a first chemical solution to be dispensed, (e) moving a predetermined volume of said first chemical solution through said opening into the mixing conduit, thereby dividing said bubble into an upstream bubble and a downstream bubble which are separated by a liquid slug containing said first chemical solution, (f) if required, introducing system liquid, thereby moving said liquid slug along the conduit to a position adjacent to the opening of a second liquid channel, (g) moving a predetermined volume of a second chemical solution into the mixing conduit causing it to mix with said liquid slug containing said first chemical solution (h) repeating steps (f) and (g) until all the desired chemical solutions have been added to the liquid slug.
2. A system comprising an apparatus and a method of calibrating said apparatus, where the apparatus comprises the features of claim 1 supplemented by a detector stationed adjacent to said mixing conduit but downstream of said openings, and the method comprises the steps (a) to (d) of claim 1 followed by the following additional steps: (a) transitorily operating said device for moving fluids in a first liquid channel thereby moving an initially unknown volume of a first chemical solution out of the opening and into the mixing conduit to form a liquid slug, thereby dividing said bubble into an upstream bubble and a downstream bubble, (b) moving canier liquid into the mixing conduit, thereby moving said slug with unknown volume until said detector detects the transition from the downstream bubble to the liquid slug, (c) continuing to move carrier liquid into the mixing conduit until the detector detects the transition from the liquid slug to the upstream bubble, (d) using the information gained, in particular the volume of canier liquid moved during step (g), to measure or estimate the volume of said first chemical solution that was dispensed at step (e), thereby calibrating the effectiveness of the apparatus in dispensing said first chemical solution from said first liquid channel, (e) repeating steps (e) to (h) for all other liquid channels that are to be used in order to calibrate them, and (0 using the calibration information thus obtained to dispense the predetermined volumes in steps (e), (g) and (h) of claim 1.
3. A method as claimed in claim 1, in which a detector is used to determine the position of both the upstream bubble and the downstream bubble so that the liquid slug can be manipulated with greater precision.
4. An apparatus as claimed in claims 1 and 2 which has, in addition to the features described in those claims, a dispensing tip, and a nosebag that is able to move up and down said dispensing tip where the nosebag comprises an enclosure with a bore that the dispensing tip can pass through, an inner chamber, and a side-arm through which fluids can be removed.
5. A method where the liquid slug claimed in claims 1 and 3 is dispensed into a target receptacle, which comprises the following steps: (a) mixing at least one chemical solution to form a liquid slug, (b) moving the nosebag claimed in claim 4 to its lower position, (c) moving canier liquid that is downstream of the liquid slug out of a dispensing tip, into the inner chamber of the nosebag, and thence through the side-arm of the nosebag optionally to waste, (d) moving the nosebag to the upper position, (e) positioning the dispensing tip in the target receptacle, and (0 moving the liquid slug out of the dispensing tip and into the target receptacle
6. A method as claimed in claim S where one or both of the upstream and downstream bubbles are moved out of the dispensing tip, into the inner chamber of the nosebag, and thence through the side-arm of the nosebag.
7. A method as claimed in claim 5 where the liquid that is upstream of the upstream bubble is moved out of the dispensing tip, into the inner chamber of the nosebag, and thence through the side-arm of the nosebag.
8. A method as claimed in any previous claim where a detector is used to find the position of the upstream and downstream bubbles prior to dispensing the liquid slug.
9. An apparatus as claimed in any previous claim in which the liquid and gas channels have inlets that are arranged along a line or lines that are at right-angles to the mixing conduit.
10. An apparatus as claimed in any previous claim in which one or more liquid channels possess constrictions at their openings where they join the mixing conduit.