WO2005094976A1

WO2005094976A1 - Microfluidic mixing

Info

Publication number: WO2005094976A1
Application number: PCT/SE2005/000403
Authority: WO
Inventors: Gerald Jesson; Mats Holmquist; Matthew Fielden
Original assignee: Gyros Patent Ab
Priority date: 2004-03-31
Filing date: 2005-03-21
Publication date: 2005-10-13
Also published as: EP1729874A1; ATE440658T1; DE602005016228D1; EP1729874B1

Abstract

A method for mixing two or more aliquots of material, said material being liquid for each of the aliquots except one that may be liquid or a solid material that is dispersed or dissolved in the resulting mixed aliquot, said mixing taking place in a microfluidic mixing unit (100,200,300). The mixing unit comprises: A) a premixing microcavity (103,203,303) that in the upstream direction is connected to said inlet arrangement, and B) a mixing microconduit (104,204,304), and C) an inlet subunit (101,201,301) that in the downstream direction is connected to said premixing microcavity (103,203,303) and/or said mixing microconduit (104,204,304). The method comprises the steps of: i) providing said unit (100,200,300), ii) providing said two or more aliquots in the premixing microcavity (103,203,303), iii) mixing said aliquots in the mixing microconduit (104,204,304), and iv) collecting said resulting mixed aliquot. The characteristic feature is: (A) the mixing unit comprises a vent (111,211,311) in a remote part or end of microconduit (104,204,304), and (B) step (iii) comprises the substeps of: (iii.a) moving said aliquots into said microconduit (104,204,304), and (iii.b) moving said aliquots (104,204,304) in the opposite direction in said microconduit (104,204,304).

Description

MICROFLUIDIC MIXING TECHNICAL FIELD AND BACKGROUND PUBLICATIONS

The present invention relates to a new method for mixing two or more aliquots of material in a mixing unit of a microchannel structure of a micro fluidic device.

The term "material" contemplates that all the aliquots are in liquid form except one of them that either is in liquid form or in a solid or semisolid form that is soluble or dispersible (=suspensible) in the liquid with which it is to be mixed. In this context solid include also semisolid materials such as gels, cells etc that are more or less soft. The liquid aliquot obtained by the innovative mixing is homogeneous and in the form of a mixture/solution or a dispersion (= suspension).

During the advent of the micro fluidic era mixing of liquid aliquots in microchannel structures primarily was accomplished by creating turbulence. However, miniaturisation led to smaller and smaller cross-sectional dimensions rendering mixing by turbulence complicated.

Mixing variants were developed that utilized mixing units that had two inlet microconduits that merged into a mixing microconduit that ended in a microcavity or chamber for collecting the resulting mixed aliquot. Mixing started by introducing separate aliquots that were transported "in parallel" in the inlet microconduits. Downstream the junction of the inlet microconduits, the two aliquots were flowing in a laminar manner in contact with each other. Mixing was accomplished by diffusion between the aliquots, i.e. a slow exchange of molecules. The mixing microconduit speeded up the process since it provided a much larger contact surface between the aliquots compared to compact structures such as microchambers. Extended mixing microconduits, however, were required which is contrary to the general trend in micro fluidics that aims at placing the largest possible number of microchannel structures in the smallest possible area. This mixing principle has been applied to circular devices in which liquid transport is driven by centrifugal force created by spinning the device around its centre. See US 6,582,663; and WO 00079285 (Tecan Trading). This centrifugally based variant was later modified to allow for improved mixing by transporting the aliquots forth and back in the mixing microconduit (radially outwards and inwards). To accomplish this air ballast chambers were included at the end of the mixing microconduit (proximal to the circumference of the device) and used to build up over-pressure during outward transportation (spinning). When spinning was stopped over-pressure was released by moving the liquid back (inwards) in the mixing microconduit. See US 6,527,432; US 20020097632; US 20030152491; and WO 01087487 (Tecan Trading).

Forth and back mixing of minute aliquots abutting each other in a microconduit has also been suggested for non-centrifugal systems. See US 6,379,929 (University of Michigan).

These earlier mixing designs were primarily adapted for relatively large volumes, typically > 1 μl such as > 5 μl, i.e. a format where wicking and/or evaporation is relatively easy to deal with. If going down in volume, e.g. into the nl-range (i.e. < 5,000 nl such as < 1,000 nl), it will become more critical to deal with this problem. Long mixing microconduits would also provide more inner surfaces meaning that relatively more material could adhere to the inner walls after emptying and contaminate subsequently introduced liquids.

Recently we published a centrifugally based mixing unit that utilized a shortened mixing microconduit in which it was possible to a) obtain enhanced mixing, b) shorten the period of time needed for a given flow rate, volume etc, c) provide a more compact mixing unit d) etc. See WO 02074438 and WO 03024598 (Gyros AB). This mixing unit comprised a precollecting or premixing microcavity between an inlet subunit and the mixing microconduit. The mixing microconduit could have alternating larger and smaller cross-sectional areas and ended in a microcavity for collecting or retaining the resulting mixed aliquot. The unit was primarily designed for mixing aliquots in the nl- range.

Transport of liquid by wicking and/or capillarity in one direction and by centrifugal force in the opposite direction in a microconduit of a micro fluidic device has previously been suggested. See WO 99058245 (Amersham Pharmacia Biotech AB) and WO 02074438 (unit 5, figure 6) (Gyros AB). Mixing of liquid aliquots was not suggested. All patent applications and issued patents referred to herein are incorporated in their entirety by reference.

OBJECTS A first object. is to provide a method for mixing in microfluidic devices that is quicker and more effective than earlier methods.

A second object is to provide a method for microfluidic mixing that circumvent the above-mentioned problems with earlier methods.

A third object is to provide a microfluidic mixing unit in which the objects related to the method can be met.

A fourth object is to provide microfluidic mixing units that can be made more compact compared to previously known microfluidic mixing units. Subobjects are mixing units that may be devoid of subunits such as a separate mixing microconduit, a collecting microcavity at the end of the mixing microconduit, an air ballast chamber connected to the end of the mixing microconduit for creating over-pressure, etc.

A fifth object is to provide microfluidic mixing methods and units that aim at mixing involving one, two or more liquid aliquots that are in the nl-range, i.e. < 5,000 nl, such as < 1,000 or < 500 nl or < 100 nl or < 50 nl. A subobject is to provide a method for dissolving and/or dispensing an aliquot of a soluble and/or dispersible solid material with a liquid aliquot within a microfluidic device.

DRAWINGS

Figure 1: This figure illustrates a mixing unit (100) in which the inlet subunit (101) comprises two inlet microconduits (102a-b) attached to a premixing microcavity (103). One of the inlet microconduit (102a) functions as a mixing microconduit (104).

Figure 2a-b: These figures illustrate a mixing unit (200) in which the inlet subunit (201) comprises a separate inlet microconduit (202) that is communicating with the premixing microcavity (203) via the mixing microconduit (204). The premixing microcavity (203) has an outlet microconduit (205) in the form of an overflow microconduit attached to its upper part. Figure 3: This figure illustrates a mixing unit (300) in which the premixing microcavity (303) is designed with a outlet microconduit (309) at its lower part for transporting a resulting mixed liquid aliquot further downstream into a microchannel structure (307).

The first digit in the reference numbers refers to the number of the relevant figure. The second and third digits to various details. Further details about the designs shown are given in WO 02074438 (Gyros AB) and WO 03024598 (Gyros AB).

Figures 1-3 also illustrate that the inlet subunit (101,201,301) may be linked to two kinds of inlet arrangement - one (108,208,308) that is common for several microchannel (107,207a,b..,307) structures and another one (110,310) that is only linked to one microchannel structure (107).

All the structures given in the drawings are primarily designed to permit liquid transport by spinning around a spin axis. This spin-driven transport may take place in the main flow path of a microchannel structure (107, 207a-c,307) or in main parts thereof such as from the mixing microconduit (104,204,304) into the premixing microcavity

(103,203,303). More details are given below. The arrow (115,215,315) shows the radial direction towards spin axis (typically the centre of a disc).

In the variants showed in the drawings: the depth in the microconduits, such as the mixing microconduits (104,204,304), is typically 100 μm and in the premixing microcavities (103,203,303) 200 μm. The width in microconduits is typically about 100-500μm.

INVENTION The present inventors have accomplished mixing according to the objects by the use of a microfluidic mixing unit comprising: a premixing microcavity (103,203,303) that is capable of containing simultaneously the aliquots to be mixed, (a) a mixing microconduit (104,204,304), and (b) an inlet subunit (101,201,301) that in the downstream direction is connected to the premixing microcavity (103,203,303) and/or to one or more mixing microconduits (104,204,304), and Two key features are (i) a vent function (111,211,311) in a part or end of the mixing microconduit (104,204,304) that is remote from the premixing microcavity, and (ii) moving the aliquots back and forth in the mixing microconduit (104,204,304).

If the remote part or end of the mixing microconduit does not contain a vent function over-pressure would be created once liquid transport start in the direction away from the premixing microcavity. This would hamper the mixing function. Without vent function the remote part would be a dead end. .

The inventors have accomplished rapid and efficient mixing in extremely short mixing microconduits (104,204,304) by transporting the aliquots out from the premixing microcavity (103,203,303) to the mixing microconduit (104,204,304) and back into the premixing microcavity (103,203,303) and repeating this back and forth transport a number of times. The preferred transport out from the premixing microcavity (103,203,303) has been by surface forces, i.e. passive transport, and in the opposite direction by centrifugal force (spinning). The inventors have further found that an inlet micoconduit (102a,302) in the inlet subunit (101,301) can be used as a mixing microconduit (104, 304) which means that there is no imperative need for a separate mixing microconduit. Back and forth transport of the aliquots to be mixed is enabled without air ballast chambers. Very compact and simple microfluidic mixing units could thus be designed. See the variants shown in Ωgures 1-3.

Initially the aliquots are more or less layered in the premixing microcavity (103,203,303). In the case the aliquots are liquid it is believed that they are transported into the mixing conduit (104,204,304) in the form of a thin film comprising parallel layers emanating from the different liquid aliquots. The thin film transport means unusually short diffusion distances and therefore a quicker and more efficient mixing. In the case one of the aliquot is a material that is soluble or dispersible, it is believed that the transport of liquid from and to the premixing microcavity will speed up dissolution and dispersion. Other mixing mechanisms may also be involved.

FIRST ASPECT: A METHOD FOR MICROFLUIDIC MIXING. This aspect of the invention thus is a method for mixing two or more aliquots of material in a mixing unit (100,200,300) of a microchannel structure I (107,207a,307) of a microfluidic device. The method comprises the steps of:

(i) providing the microfluidic device comprising the mixing unit (100,200,300) in which there are a) a premixing microcavity (103,203,303), b) a mixing microconduit (104,204,304), and c) an inlet subunit (101,201,301) that in the downstream direction is connected to the premixing microcavity (103,203,303) and/or the mixing microconduit (104,204,304), (ii) providing said aliquots in said premixing microcavity (103,203,303), (iii) mixing said aliquots by moving them from the premixing microcavity (103,203,303) to the mixing microconduit (104,204,304), and (iv) collecting the resulting aliquot.

The main characteristic features of the method is that (A) the mixing unit (100,200,300) has a vent function (111,211,311) in the end of the mixing microconduit that is remote from the premixing microconduit (in the dead end), and (B) step (iii) comprises moving the aliquots within the mixing microconduit (104,204,304) a) from the premixing microcavity (103,203,303) (step iii. a), and b) back towards the premixing microcavity (103,203,303) (step iii.b). Optionally the sequence "step (iii.b) followed by step (iii.b)" may be repeated, for instance once, twice, thrice or more times, for instance 1-5 times, such as 1-10 times or 1-50 times or 1 - 100 times.

If not otherwise apparent from the context, terms saying that units/subunits/functions are connected to each other or are communicating with each other contemplates that liquid shall be transported between them. The units/subunits/functions thus are in fluid communication/fluidly connected to each other.

Step (i): The mixing unit If not otherwise apparent from the context, the terms "upper" and "higher" versus "lower", "upward" versus "downward", "inward" versus "outward", "above" versus "lower" etc with respect to positions on the device refer to relative locations in relation to the direction of the main force used to drive liquid transport or flow downstream within the major parts of a microchannel structure (107,207a-c,307), for instance within the major flow path. This in particular applies to forces used for overcoming passive valve functions (111,112,113,214,216,311,312,313,317) if such valves are present. If liquid transport or flow in the main flow path is driven by centrifugal force caused by spinning the device about a spin axis (110,210,310), then the following applies: a) upper and higher levels/positions are closer to the spin axis than lower levels/positions, b) upward and inward mean towards the spin axis or from a lower radial position to a higher radial position relative to the spin axis, c) downward and outward is the opposite to the direction given in (b), etc. Downstream and upstream on the other hand refer to the process stream carried out in a microchannel structure, i.e. an upstream position/step is placed/carried out before a downstream position.

The inlet subunit (101,201,301) typically comprises one, two or more inlet microconduits (102a-b,202,302a-b) each of which in the upstream direction is communicating with an inlet arrangement (108,110,208,308,310) of microchannel structure I (107,207 a,307). This kind of inlet arrangement may be separate (110,310) for a microchannel structure I, or alternatively be common (108,208,308) to a microchannel structure I (107,207a,307) and one or more additional microchannel structures (207b-c, not shown in figures 1 and 3) that are present on the microfluidic device. These microchannel structures may be microfluidically equivalent (207b-c) to microchhannel structure I, for instance. The inlet subunit (101,201,301) of the mixing unit (100,200,300) may thus be connected to either one, two or more separate and/or one, two or more common inlet arrangements [(110,310) and (108,208,308), respectively]. Intermediate between an inlet micronduit (102a-b,202,302a-b) and an inlet arrangement (110,108,208,310,308) there may be other microfluidic functionalities, e.g. for performing reactions, separations, detections etc. See further under the heading "General features of microfluidic devices".

One, two or more of the inlet microconduits (102a,302a) may in certain variants of the invention also function as a mixing microconduit (104,304) as represented in figure 1 and figure 3.

The inlet subunit (101,201,301) is typically connected to the upper part and/or the upstream end of the premixing microcavity (103,303) as suggested in the variant represented by figure 1 and figure 3, and/or to the mixing microconduits (204) as represented by figure 2. The connection to the premixing microcavity or to the mixing microconduit is typically via the above-mentioned inlet microconduit(s).

The premixing microcavity (103,203,303) is capable of simultaneously containing the aliquots to be mixed in the mixing microconduit (104,204,304). In preferred variants this means that the total volume of aliquots to be mixed is less than the volume of the premixing microcavity (103,203,303). In a less preferred variant a part of the premixing microcavity (103,203,303) coincides with a part of the mixing microconduit (104,204,304). For instance if the total volume of the aliquots to be mixed is larger than the volume of the rounded premixing vessel in figures 1-3, liquid will also fill a part of the mixing microconduit. The part of the mixing microconduit that is closest to the rounded vessel will then also be part of the premixing microcavity.

The premixing microcavity (103,203,303) may comprise an outlet end at which there is connected one or more outlet microconduits (205,309) for transporting the mixed aliquot downstream into the microchannel structure as illustrated in figures 2 and 3. An outlet microconduit (205,309) may lead to a waste function/microconduit (218,318) or to a unit (319) in which further processing may take place, such as a reaction, separation, etc. An outlet microconduit (205,309) may be present in the downstream end of the premixing microcavity (203,303) and may be placed at about the same level (205) as, or beneath (309) or above the junction between the premixing microcavity (103,203,303) and the inlet subunit (101,201,301). See figures 2 and 3. The premixing microcavity may have the inlet subunit and an outlet microconduit (205) connected to its upper part. The outlet microconduit (205) may then function as an over- flow microconduit and the premixing microonduit (203) as a volume-metering microcavity for a mixed aliquot. In the case a second outlet microconduit is connected to the lower part of the premixing microcavity this latter outlet microconduit may be used for transporting a metered mixed liquid aliquot downstream into the microchannel structure for further processing (not shown).

There are one, two or more mixing microconduits connected to the premixing microcavity (103,203,303). In total the inner volume of the mixing microconduit(s) (104,204,304) should be capable of simultaneously containing all the aliquots to be mixed. If there is only one mixing microconduit (104,204,304) this means that its volume in preferred variants is equal to or larger than the sum of the volumes of the aliquots to be mixed. In less preferred variants the volume of the mixing microconduit(s) (104,204,304) may be less than the total volume of these aliquots. In these latter variants the aliquots will only be partially transported into the mixing microconduit during each forth and back cycle which most likely will require larger number of cycles and/or slower transport in each cycle for efficient mixing.

The connection between the mixing microconduit (104,204,304) and the premixing microcavity (103,203,303) may be at about the same level as, or below or above the connection between the inlet subunit (101,201,301) and the premixing microcavity (103,203,303). The proper choice will depend on the design of the mixing unit (100,200,300) and/or the forces used for transporting the aliquots forth and back between the premixing microcavity (103,203,303) and the mixing microconduit (104,204,304), among others. The forth and back transport preferably utilizes passive transport in one direction, preferably in the direction from the premixing microcavity. Passive transport is then preferably combined with centrifugal force in the opposite direction. In alternative variants centrifugal force or passive transport may be combined with other forces for transporting liquid in the opposite direction. One can also envisage variants in which neither centrifugal force nor passive transport is used.

If passive transport is utilized the chemical and geometrical/physical surface characteristics of the inner walls of the mixing microconduit and the premixing microcavity should be designed such that surface forces, such as capillarity and/or wicking, gives the desired transport. Passive transport from the premixing microcavity (103,203,303) to the mixing microconduit (104,204,304) can be arranged if the latter a) is hydrophilic compared to the premixing microcavity (103,203,303) and/or b) has one, two or more length-going inner edges stretching into the premixing microcavity (103,203,303) and/or c) has at least one cross-sectional dimension that is less than the corresponding cross- sectional dimension in the premixing microcavity (103,203,303), and/or d) has inner walls with a wettability that is higher that the wettability of the inner walls of the premixing microcavity (103,203,303), etc.

A hydrophilic mixing microconduit (104,204,304) contemplates that the conduit will be at least partially filled by self-suction (passive transport) once the front of liquid has passed over the junction between the premixing microcavity (103,203,303) and the mixing microconduit (104,204,304). In the case the premixing microcavity is more hydrophilic than the mixing microconduit self-suction is from the mixing microconduit to the premixing microcavity with active liquid transport in the reverse direction, e.g. by centrifugal force/spinning.

Length-going inner edges are defined by intersecting inner walls of a microconduit. See figure 1 of WO 02074438 (Gyros AB). Inner edges promote transport by wicking and typically stretch from the mixing microconduit into the premixing microcavity.

For item (c), the term "less" typically means that the depth and/or the width of the mixing microconduit (104,204,304) is/are < 0.75, such as < 0.5 or < 0.25 times the corresponding dimension in the premixing microcavity (103,203,303) at the junction between these two subunits. In the case the depth and/or the width of the mixing microconduit (104,204,304) is/are larger than the corresponding dimension in the premixing microcavity (103,203,303) this would promote passive transport in the other direction, i.e. from the mixing microconduit to the premixing microcavity.

Higher wettability in context (d) primarily means that inner walls of the mixing microconduit (104,204,304) have lower water contact angles than inner walls of the premixing microconduit (103,203,303), typically with water contact angles < 50°, such as < 35° or < 20° or < 5°. The mixing microconduit (104,204,304) thus may comprise inner walls with a water contact angle < 90° and the premixing microcavity (103,203,303) inner walls with a water contact angle > 90°. Further details about combining inner walls of different wettabilities in the individual subunits are given elsewhere in this specification. By permitting inner walls of premixing microcavities

(103.203.303) to have higher wettabilities than inner walls of the mixing microconduit

(104.204.304) passive liquid transport in the reverse direction can be promoted, i.e. from the mixing microconduit to the premixing microcavity.

A similar effect may potentially also be accomplished if at least a section of the mixing microconduit comprises a plurality of thinner microconduits/ pores, for instance. See figure 2 [and the discussion about unit 5 (figure 6) in WO 02074438 (Gyros AB)].

One can also envisage alternative designs that are based on what has been outlined in US 20030152491 (Tecan Trading) but with vent function placed in the air ballast chambers. In these designs the mixing microconduit contains inner walls that have a) an increasing non-wettability in the direction out from the premixing microcavity, and/or b) an increasing surface to volume ratio in the same direction. Both (a) and (b) may be designed to promote passive transport towards the premixing microcavity. As outlined in US 200300152491 these variants are adapted for moving liquid from the premixing microcavity into the mixing microconduit by centrifugal force.

A vent function (111,211,311) in a remote part or end of the mixing microconduit (104,204,304) vents out gas over-pressure that may be created by transport of liquid within the mixing microconduit. Preferably the mixing unit also has vent functions (122,222,322) in air traps, i.e. in positions in which air may collect without preventing liquid transport through the unit/subunit. Venting is typically either directly or indirectly to ambient atmosphere. There are two main kinds of vent functions a) vent functions that are purely used for venting in and/or out gas such as air (122,211,222,322), and b) vent functions that are used both as gas vents and as liquid inlets and/or liquid outlets (e.g. 111,214,311). A vent function in a mixing microconduit (104,204,304) is typically placed in the part that is most remote from the premixing microcavity (103,203,303), if possibly in an upper end. The mixing unit (100,200,300) is typically delineated towards other parts of the microchannel structure by anti-wicking functions. The reason is to avoid losses by undesired transport of liquid due to wicking. These anti-wicking functions are typically in the form of local changes or breaks in chemical and/or geometric surface characteristics, for instance as described in WO 02074438 (Gyros AB). Abrupt changes are more preferred than smooth changes. Anti-wicking functions may thus be present in a) the end of the mixing microconduit (104,204,304) that is remote from the premixing microcavity (i.e. 111,211,311), b) the inlet microconduit(s) (102,202,302) used for introducing liquid into the premixing microcavity (i.e. 111,112,214,311,312), c) the outlet microconduit(s) (205,309) used for transporting liquids into parts of the microchannel structure that are downstream the microfluidic mixing unit (i.e. 216,317). The anti-wicking function associated with a junction between a microconduit and a microcavity are often associated with a dimension change.

Each of the anti-wicking functions discussed in the previous paragraph a) may coincide with a valve function that for instance is used to control the transport of liquid aliquots into or out of the premixing microcavity, or b) may be a pure vent function, for instance in a remoter part or end of the mixing microconduit or in an air trap.

Step (ii): Providing the aliquots to be mixed The liquid aliquots are typically introduced via the inlet subunit (101,201,301).

An aliquot of solid material is preferably introduced via the inlet subunit as one or more liquid aliquots (as a solution or dispersion) and subsequently dried or desiccated in the premixing microcavity (103,203,303). The starting liquid aliquot(s) may or may not contain an agent stabilizing the solid components during drying/desiccation and/or subsequent storage and mixing. This may be carried out as disclosed in copending International Application WO 2004083108 (Gyros AB). The solid material may alternatively be introduced during the manufacture of the microfluidic device, for instance before attaching a lid to a substrate exposing uncovered forms of microchannel structure I and other microchannel structures of the device. See below.

The desiccated/dry material may comprise buffer substances, salts, reactants, reagents, analytes etc required for processing the resulting mixed liquid aliquot according to a predetermined protocol.

The liquid aliquots contemplated are in most cases aqueous. Their surface tension is typically < 30 mN/m or <25 mN/m, such as from 10 mN/m and upwards.

Step (iii): Mixing by moving liquid forth and back

This movement starts by transporting the aliquots out from the premixing microcavity

(103.203.303) into the mixing microconduit (104,204,304). Once this has been done the forth and back transport may take place solely in the mixing microconduit (104,204,304) although it is preferred to end each cycle with placing the aliquots in the premixing microcavity (103,203,303).

In advantageous variants of the invention, centrifugal force created by spinning the device about a spin axis is utilized either for liquid transport in the direction away from the premixing microcavity or towards the premixing microcavity. Passive transport, such as by capillary force and/or wicking, is then used for transport in the opposite direction.

In the most advantageous variants passive transport is used for transport from the premixing microcavity (103,203,303) to a part of the mixing microconduit

(104.204.304) that is remote from the premixing microcavity (103,203,303). The remote part is typically above the premixing microcavity (103,203,303), e.g. at a shorter radial distance than the junction between the mixing microconduit and the premixing microcavity if spinning is used for reversing the transport.

Other forces may also be used to carry out the cycliing transport (repetitive forth and back transport) within the mixing microconduit. Micropumps, for instance, can be used to move a liquid aliquot in any desired direction required by a particular configuration of a premixing microcavity and a mixing microconduit. This may require other designs and configurations of the subunits of the mixing unit of the invention.

Step (iv): Collecting of the resulting aliquot (mixed aliquot) This step typically means that the resulting mixed aliquot is collected in the premixing microcavity (103,203,303). Further processing may take place in the premixing microcavity or in a reaction microcavity (319) downstream the premixing microcavity. Downstream transport may take place via a separate outlet microconduit (205,309) as discussed above. One can also envisage downstream transport via the mixing microconduit or via an inlet microconduit of the inlet subunit (not shown).

In certain variants downstream processing or at least a part of it may take place in a mixing microconduit (104,204,304) or in an inlet subunit (102,202,302). Detection, for instance, of the outcome of the result of a reaction taking place during mixing or in the mixed aliquot, monitoring of the mixing etc may be performed in the mixing microconduit or in the inlet subunit.

Transport of liquid aliquots between the premixing microcavity and subunits other than the mixing microconduit. Centrifugal force (spinning about a spin axis) and/or capillary force represent(s) the most attractive ways for the inventors for transporting liquid aliquots from an inlet subunit (102,202,302) to the premixing microcavity (103,203,303) and from the premixing microcavity to downstream parts of a microchannel structure, e.g. via the outlet microconduit (205,309). In preferred variants one utilizes a microfludic device that is associated with a spin axis that is common for utilizing centrifugal force for two or three of the following transports a) one direction in the mixing microconduit, with preference for towards the premixing microcavity b) from the inlet subunit to the premixing microcavity and c) from the premixing microcavity via the outlet microconduit to downstream parts of the microchannel structure. This means that the most downstream part of the inlet subunit (101,201,301) should be closer to the spin axis used than the premixing microcavity (103,203,303). It also means that the inlet subunit, such as its inlet microconduits (102a-b,202,302a-b) (if present), should be connected to the upper part of the premixing microcavity (103,203,303). Similarly an outlet microconduit (205,309) should transport liquid collected in the premixing microcavity (303) to a downstream position that is at a lower level than the premixing microcavity (303), i.e. to a position that is more remote from the spin axis than the premixing microcavity (303). The junction between the outlet microconduit (205) and the premixing microcavity (203) may be at an upper part of the premixing microcavity, for instance if the outlet microconduit is simply used as an overflow channel. If the main part of the resulting mixed aliquot is to be transported downstream via the outlet microconduit (309) the junction should be in the lower part of the premixing microcavity (303).

Other combinations of forces may also be used as suggested for the forth and back transport in the mixing microconduit and for microfluidic devices in general.

General features of microfluidic devices.

A microfluidic device comprises one or more microchannel structures in which liquid flow is used for transporting and processing liquid aliquots containing various kinds of reactants, analytes, products, samples, buffers and/or the like. The volumes of the aliquots are typically in the nanolitre (nl) range. Each microchannel structure comprises all the functionalities needed for performing the experiment that is to be performed within the microfluidic device. The microchannel structure contains one or more microcavities and/or microconduits that have a cross-sectional dimension that is < 10³ μm, preferably < 5 x 10 μm, such as < 10 μm. The nl-range has an upper limit of 5,000 nl. In most cases it relates to volumes < 1,000 nl, such as < 500 nl or < 100 nl.

A microchannel structure thus may comprise one, two, three or more functional parts selected amongst: a) inlet arrangements comprising for instance one or more inlet ports/inlet openings, possibly together with a volume-metering microcavity, b) microconduits for liquid transport, c) reaction microcavities/units; d) mixing units, for instance according to the present invention; e) units for separating particulate matters from liquids (may be present in the inlet arrangement), f) units for separating dissolved or dispersed/suspended components in the sample from each other, for instance by capillary electrophoresis, chromatography and the 5 like; g) detection microcavities/units; h) waste conduits/microcavities/units; i) valves; j) vents to ambient atmosphere; 10 k) anti-wicking functions; 1) liquid directing functions etc. A functional part may have two or more functionalities: 1. a reaction microcavity and a detection microcavity may coincide, 2. a volume-metering function may comprise one or more valve functions and a 15 metering microcavity and/or an anti-wicking function, 3. a reaction microcavity may comprise one or more valve functions and/or anti- wicking functions, 4. a passive valve function based on a non-wettable surface break may comprise also an anti-wicking function etc.

20 A mixing unit of the invention may have a mixing microconduit (104,204) that also function as an inlet microconduit (102a,302a). Various kinds of functional units in microfluidic devices have been described by Gyros AB/Amersham Pharmacia Biotech AB: WO 9955827, WO 9958245, WO 02074438, WO 0275312, WO 03018198, WO 03024598 and by Tecan/Gamera Biosciences: WO 0187487, WO 0187486, WO

25 0079285, WO 0078455, WO 0069560, WO 9807019, WO 9853311.

An inlet arrangement (110,108,208,310,308) typically comprises an inlet port (123,224a-b,323,324) and at least one volume-metering microcavity (125,126, 226a- c,325,326). In an advantageous variant, there is one separate inlet arrangement 30 (110,310) per microchannel structure (107,207a-c,307) and mixing unit (100,200,300). In another advantageous variant, there is an inlet arrangement (108,208,308) that is common to all or a subset (206) of the microchannel structures of the device and comprises a common inlet port (224a-b,324) and a distribution manifold with one volume-metering microcavity (126,226,326) for each microchannel structure/mixing unit (107,207a-c,307/l 00,200,300) of the subset (206). In both variants, each of the volume-metering microcavities (125,126,226,325,326) in turn is communicating with downstream parts of its microchannel structure, e.g. the mixing unit. MicroChannel structures linked together by a common inlet arrangement and/or common distribution manifold define a subset/subgroup of the microchannel structures of the device. Each volume-metering cavity (125,126,226,325,326) typically has a valve (111,112;214;311,312) at its outlet end. This valve is typically passive, for instance utilizing a change in chemical surface characteristics at the outlet end, such as a boundary between a hydrophilic and hydrophobic surface (hydrophobic surface break) (WO 99058245 (Amersham Pharmacia Biotech AB)) and/or in geometric/physical surface characteristics (WO 98007019 (Gamera)). See also WO 02074438 (Gyros AB), WO 04103890 (Gyros AB) and WO 04103891 (Gyros AB) for preferred valves that are based on hydrophobic surface breaks.

Typical inlet arrangements with inlet ports, volume-metering microcavities, distribution manifolds, valves etc have been presented in WO 02074438 (Gyros AB), WO 02075312 (Gyros AB), WO 02075775 (Gyros AB) and WO 02075776 (Gyros AB).

The microfluidic device may also comprise other common microchannels/ microconduits that connect different microchannel structures. Common channels/ conduits including their various parts such as inlet ports, outlet ports, vents, etc., are considered part of each of the microchannel structures they are common for.

Each microchannel structure has at least one inlet opening for liquids and at least one outlet opening for excess of air (vents) and possibly also for liquids.

The number of microchannel structures/device is typically > 10, e.g. > 25 or ≥ 90 or > 180 or > 270 or > 360. At least one, preferably two or more such as all or a subset thereof, comprises the mixing unit of the invention.

The microchannel structures of a device are arranged such that at least mixing according to the invention can be carried out in a timely parallel fashion for at least two or more or one or more subgroups of the microchannel structures of the same microfluidic device. A subgroup in this context comprises microchannel structures linked together by a common functionality such as a common inlet arrangement, which for instance is common for 4-25 microchannel structures. MicroChannel structures in a subgroup are typically functionally equivalent, i.e. they can be used in a timely parallel fashion at least with respect to mixing in the mixing microconduit (104,204,304).

Different principles may be utilized for transporting the liquid within the microfluidic device/microchannel structures between two or more of the functional parts described above. Inertia force may be used, for instance by spinning the disc as discussed in the subsequent paragraph. Other useful forces are electrokinetic forces, non-electrokinetic forces such as capillary forces, hydrostatic pressure etc.

A microfluidic device typically is in the form of a disc. The preferred formats have an axis of symmetry (C_n) that is perpendicular to or coincides with the disc plane. In the former case n is an integer > 2, 3, 4 or 5, preferably ∞ (C_∞). In the latter case n is typically 2. In other words the disc may be rectangular, such as in the form of a square, or have other polygonal forms. It may also be circular. Once the proper disc format has been selected centrifugal force may be used for driving liquid flow, e.g. by spinning the device about a spin axis that typically is perpendicular to or parallel with the disc plane. Parallel in this context includes that the spin axis coincides with the disc plane. In the most obvious variants at the priority date, the spin axis coincides with the above- mentioned axis of symmetry. Preferred variants in which the spin axis is not perpendicular to the disc plane are given in International Patent Application WO 04050247 (Gyros AB)

For preferred centrifugal-based variants, each microchannel structure comprises an upstream section that is at a shorter radial distance than a downstream section relative to the spin axis. Spinning of the device about this spin axis will then induce transportation of liquid from the upstream section to the downstream section.

The preferred devices are typically disc-shaped with sizes and forms similar to the conventional CD-format, e.g. sizes that corresponds CD-radii that are the interval 10% - 300 % of the conventional CD-radii (about 12 cm). The upper and/or lower sides of the disc may or may not be planar. Microchannels/microcavities of a microfluidic device may be manufactured from an essentially planar substrate surface that exhibits the channels/cavities in uncovered form that in a subsequent step are covered by another essentially planar substrate (lid). See WO 91016966 (Pharmacia Biotech AB) and WO 01054810 (Gyros AB). Both substrates are preferably fabricated from plastic material, e.g. plastic polymeric material.

The fouling activity and hydrophilicity of inner surfaces should be balanced in relation to the application. See for instance WO 0147637 (Gyros AB).

The terms "wettable" and "non-wettable" with respect to inner walls contemplate that the inner surface of an inner wall has a water contact angle < 90° or > 90°, respectively. In order to facilitate efficient transport of a liquid between different functional parts, inner surfaces of the individual parts should primarily be wettable, preferably with a contact angle < 60° such as < 50° or < 40° or < 30° or < 20°. These wettability values apply for at least one, two, three or four of the inner walls of a microconduit. In the case one or more of the inner walls have a higher water contact angle, for instance by being essentially non-wettable, this can be compensated for by a lower water contact angle for the other inner wall(s). The wettability, in particular in inlet arrangements should be adapted such that an aqueous liquid will be able to fill up an intended microcavity/microconduit by capillarity (self suction) once the liquid has started to enter the cavity, typically with the inner surfaces being in a dry state. A hydrophilic inner surface in a microchannel structure may comprise one or more local hydrophobic surface breaks in a hydrophilic inner wall, for instance as part of a passive valve, an anti-wicking function, a vent solely functioning as a vent to ambient atmosphere etc (rectangles in figure 1). See also WO 99058245 (Gyros AB) and WO 02074438 (Gyros AB), and WO 04103890 (Gyros AB) and WO 04103891 (Gyros AB) for preferred hydrophobic surface breaks.

Contact angles refer to values at the temperature of use, typically +25°C, are static and can be measured by the method illustrated in WO 00056808 (Gyros AB) and WO 01047637 (Gyros AB). SECOND ASPECT: MIXING UNIT AND MICROCHANNWEL STRUCTURE OR MICROFLUIDIC DEVICE COMPRISING THE MIXING UNIT.

A second aspect of the invention is the mixing unit discussed above possibly as being part of a microchannel structure/micro fluidic device. One of the main characteristic features is that one or two cross dimension (depth and/or width) of the mixing microconduit (104,204,304) is smaller compared to the corresponding cross dimension in the premixing microcavity (103,203,303). The ranges discussed elsewhere in this specification applies. The second aspect of the invention alternatively comprises other unique features of the mixing unit discussed herein. In subaspects characterizing features are as outlined for the mixing unit used in the first aspect (method)

Certain innovative aspects of the invention are defined in more detail in the appending claims. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

C L A I M S

1. A method for mixing two or more aliquots of material, said material being liquid for each of the aliquots except for one that may be liquid or a solid material that is dispersed or dissolved in the resulting mixed aliquot, said mixing taking place in a mixing unit (100,200,300) of a microchannel structure I (107,207a,307) of a microfluidic device which mixing unit comprises: (A) a premixing microcavity (103,203,303), and (B) a mixing microconduit (104,204,304) connected to the premixing microcavity, and (C) an inlet subunit (101,201,301) that in the downstream direction is connected to said microcavity (103,203,303) and/or said microconduit (104,204,304), and which method comprises the steps of: (i) providing said microfluidic device, (ii) providing said two or more aliquots in said microcavity (103,203,303), (iii) mixing said aliquots in said microconduit (104,204,304), and (iv) collecting said mixed aliquot, characterized in that (A) said microconduit (104,204,304) comprises a vent function (111,211,311) to ambient atmosphere in a remote part or end of the mixing unit (100,200,300), and (B) step (iii) comprises the substeps of: (iii. a) moving said aliquots into said microconduit (104,204,304), and (iii.b) moving said aliquots in said microconduit (104,204,304) in the opposite direction.

2. The method according to claim 1, characterized in that said moving in one of steps (iii. a) and step (iii.b) is supported by passive transport, preferably in step (iii. a).

3. The method according to claim 2, characterized in that that said moving in the other one of steps (iii. a) and (iii.b) is by centrifugal force, preferably in step (iii.b).

4. The method according to any of claim 1-3, characterized in that step (iii) comprises repeating the sequence: step (iii. a) followed by step (iii.b).

5. The method according to any of claims 1-4, characterized in that step (iv) comprises collecting said mixed aliquot in the premixing microcavity (103,203,303).

6. The method according to any of claims 1-5, characterized in that said inlet subunit (101,201,301) comprises one, two or more inlet microconduits (102a-b,202,302a-b) directly connected to the premixing microcavity (103,203,303) or to the mixing microconduit (104,204,304).

7. The method according to claim 6, characterized in that said mixing microconduit (104,304) coincides with one of the inlet microconduits (102a,302a).

8. The method according to any of claims 6-7, characterized in a) that said inlet subunit (101,201,301) comprises two or more of said inlet microconduits (102a- b,302a-b), and b) that at least two of said aliquots are provided via separate inlet microconduits.

9. The method according to any of claims 6-8, characterized in that at least one of said one, two or more inlet microconduits (102a-b,202,302a-b) is connected in the upstream direction to an inlet arrangement (110,108,208,310,308) of the microchannel structure.

10. The method according to claim 9, characterized in that the inlet arrangement (110,108,208,310,308) comprises an inlet port (123,224a-b,323,324) and a volume- metering microcavity (125,126,226a-c,325,326) downstream to the inlet port.

11. The method according to any of claims 1-10, characterized in that (A) said mixing unit comprises (a) an outlet microconduit (205,309) connected to the premixing microcavity (203,303), and (b) a valve function (216,317), preferably passive, which is associated with the junction between the premixing microcavity (103,203,303) and said outlet microconduit (205,309), (B) step (iv) comprises collecting the mixed aliquot in said premixing microcavity (103,203,303), and (C) the mixed aliquot is transported further downstream in the microchannel structure via said outlet microconduit (205,309).

12. The method according to any of claims 9-11, characterized in that 5 (A) the microfluidic device comprises one or more additional microchannel structures (II, in, IN . . .) (207b-c), and (B) the inlet arrangement (108,208,308) (a) is common to microchannel structure I and at least one of said additional microchannel structures, 10 (b) has an inlet port (224a-b) that is common to microchannel structure I (207a) and at least one of said additional microchannel structures (207b- c), and (c) has one volume-metering microcavity (226a-c) per microchannel structure/mixing unit (207a-c/200a-c). 15

13. The method according to any of claims 1-12, characterized in that (a) the microfluidic device is designed for being spun about a spin axis to accomplish at least one of step (ii), step (iii. a) or step (iii.b), and step (iv), and (b) performing at least one of step (ii), step (iii. a) or step (iii.b), and step (iv) by 20 spinning the device about said spin axis.

14. The method according to any of claims 13, characterized in that (a) the microfluidic device is designed for being spun about a spin axis to accomplish step (iii.b), e.g. the section/position in the mixing microconduit

25 within/at which transport of liquid in step (iii. a) is halted is at a shorter radial distance than the section through which the liquid aliquots have passed before the halting, and (b) step (iii.b) comprises spinning the microfluidic device about said spin axis.

30 15. The method according to any of claims 13-14, characterized in that (a) the microfluidic device is designed for being spun about a common spin axis for performing steps (ii), (iii.b), (iv) and if present (iv), and (b) step (ii), (iii.b), (iv) and if present (v) comprise spinning the microfluidic device about said spin axis.

16. The method according to any of claims 13-15, characterized in that said microfluidic device has an axis of symmetry (C„, n = an integer 2, 3, 4, 5 .... ∞) that coincides with the spin axis, with preference for n > 6, such as ∞.

17. The method according to any of claims 1-16, characterized in that the microfluidic device is disc-shaped.