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Title:
METHOD OF OPERATING A FLUIDIC DEVICE AND A FLUIDIC DEVICE FOR USE IN THE METHOD
Document Type and Number:
WIPO Patent Application WO/2008/155519
Kind Code:
A1
Abstract:
A method comprises introducing a fluid carrying mobile elements, such as particles (24), into a fluidic device. Simultaneously a mobile element receiving fluid and one or more further fluids are also introduced into the fluidic device. The fluids are passed through a chamber (12) in respective, parallel fluid streams (32, 34, 36, 38, 40). The fluid streams are arranged such that each fluid stream contacts at least another one of the fluid streams in the chamber (12). The dimensions of the chamber are such that the fluid streams undergo laminar flow and remain generally distinct from one another. A force is applied to the mobile elements (24) which causes the mobile elements to leave the fluid stream (32) of the mobile element carrying fluid, pass through the fluid stream(s) (34, 36, 38) of the further fluid(s) and enter into the fluid stream (40) of the mobile element receiving fluid. As the mobile elements pass through the fluid streams reactions can take place between species bound to the mobile elements and species carried in the fluid streams.

Inventors:
ILES ALEXANDER (GB)
PAMME NICOLE (GB)
Application Number:
PCT/GB2008/001992
Publication Date:
December 24, 2008
Filing Date:
June 11, 2008
Export Citation:
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Assignee:
UNIV HULL (GB)
ILES ALEXANDER (GB)
PAMME NICOLE (GB)
International Classes:
B01L3/00; G01N33/543
Other References:
JANDIK P ET AL: "Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 954, no. 1-2, 19 April 2002 (2002-04-19), pages 33 - 40, XP004348575, ISSN: 0021-9673
WANG ET AL: "Investigation of active interface control of pressure driven two-fluid flow in microchannels", SENSORS AND ACTUATORS A, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 133, no. 2, 30 January 2007 (2007-01-30), pages 323 - 328, XP005864728, ISSN: 0924-4247
KIERAN CURRAN ET AL: "Spectral intensity mapping and analysis of dyed microflows", MICROFLUIDICS AND NANOFLUIDICS, SPRINGER, BERLIN, DE, vol. 1, no. 2, 1 May 2005 (2005-05-01), pages 146 - 154, XP019359734, ISSN: 1613-4990
Attorney, Agent or Firm:
MATHISEN MACARA & CO. (6-8 Swakeleys RoadIckenham, Uxbridge UB10 8BZ, GB)
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Claims:
CLAIMS

1. A method of operating a fluidic device comprising: introducing a fluid carrying mobile elements, a mobile element receiving fluid and one or more further fluids into a chamber; passing the fluids through the chamber in respective, parallel fluid streams wherein each fluid stream contacts at least another one of the fluid streams; and applying a force or forces to the mobile elements to cause the mobile elements to move from the fluid stream of the mobile element carrying fluid through the fluid stream or sequentially through the respective fluid streams of the one or more further fluids and then into the fluid stream of the mobile element receiving fluid.

2. A method according to claim 1, wherein each mobile element carries a first species and the or at least one of the one or more further fluids carries a second species which binds to the first species.

3. A method of operating a fluidic device comprising: introducing a fluid carrying mobile elements, and one or more further fluids, into a chamber; passing the fluids through the chamber in respective, parallel fluid streams wherein each fluid stream contacts at least another one of the fluid streams; and applying a force or forces to the mobile elements to cause the mobile elements to move from the fluid stream of the mobile element carrying fluid and into the or sequentially into each fluid stream of the one or more further fluids; wherein each mobile element carries a first species and the or at least one of the one or more further fluids carries a second species which binds to the first species.

4. A method according to claim 2 or claim 3, wherein there are at least two of said further fluids, a first one of the further fluids carrying the second species and a second one of the further fluids carrying a third species, the second species undergoing said binding to the

tirst species when the mobile elements pass through the fluid stream of the first further fluid and subsequently the third species binding to the second species already bound to the first species when the mobile elements pass through the fluid stream of the second further fluid.

5. A method according to claim 4, wherein subsequently to passing through the first further fluid and prior to passing through the second further fluid, the mobile elements pass through the fluid stream of a third further fluid which serves to minimise entry of unbound second species into the second further fluid.

6. A method according to claim 4 or claim 5, further including detecting mobile elements carrying both the second and third species.

7. A method according to claim 6, wherein the third species facilitates or allows said detection.

8. A method according to claim 7, wherein the third species is fluorescent when bound to the second species and the detection is fluorescence detection.

9. A method according to any one of claims 6 to 8, when dependent on claim 2, wherein the mobile elements carrying both the second and third species enter into the fluid stream of the mobile element receiving fluid and the mobile element receiving fluid leaves the chamber for said detection.

10. A method according to claim 9, wherein subsequently to passing through the fluid stream of the second further fluid the mobile elements carrying the second and third species pass through the fluid stream of a fourth further fluid to minimise entry of unbound third species into the mobile element receiving fluid.

11. A method according to any one of claims 2 to 10, wherein one of the first species and the second species is an antibody, and the other one is an antigen.

12. A method according to any one of claims 2 to 10, wherein one of the first species and the second species is a first single stranded nucleic acid, and the other one is a second single stranded nucleic acid which anneals to the first single stranded nucleic acid.

13. A method according to claim 4 or claim 5, wherein the second and third species form part of a synthetic molecule synthesised on the mobile elements during the method.

14. A method according to claim 4, 5 or 13, wherein the binding between the second and third species is covalent binding.

15. A method according to claim 4, 5, or 13, wherein the binding between the second and the third species is physical adsorption or ionic binding.

16. A method according to any one of claims 1 to 3, wherein a molecular recognition procedure is performed on or in the mobile elements with successive steps in the molecular recognition procedure taking place in respective ones of the fluid streams.

17. A method according to any one of claims 1 to 3, wherein a multistep molecular synthesis is performed on or in the mobile elements , with successive steps of the multistep synthesis taking place in respective ones of the fluid streams.

18. A method according to any one of claims 1 to 3, wherein the mobile elements carry a species which undergoes successive chemical modifications on or in the mobile elements, the successive modifications taking place in respective ones of the fluid streams.

19. A method according to any preceding claim, wherein the application of the force or forces comprises applying a magnetic field across the fluid streams.

20. A method according to claim 19, wherein the mobile elements are superparamagnetic particles and the force or forces comprise magnetic attraction generated during the application of the magnetic field.

21. A method according to claim 19, wherein the mobile elements are diamagnetic particles and the force or forces comprise diamagnetic repulsion generated during the application of the magnetic field.

22. A method according to any preceding claim, wherein the force or forces comprise a gravitational or centrifugal force or buoyancy.

23. A method according to any preceding claim, wherein the force or forces comprise a dieletrophoretic force, an ultrasound generated force, or a force applied by optical tweezers.

24. A method according to any preceding claim, wherein the or each of at least one of the fluids is introduced into the chamber through a plurality or a respective plurality of adjacent inlets.

25. A method according to claim 24, wherein the or each plurality of inlets is fed by a common channel which branches to join the chamber at the plurality of inlets.

26. A method according to claim 24 or claim 25, wherein the inlets of the or each plurality of inlets are arranged in a line extending across the width of the corresponding flow stream.

27. A method according to claim 1 or any claim dependent thereon, wherein the mobile element receiving fluid leaves the chamber via an outlet which leads to a detector.

28. A method according to any preceding claim, wherein the mobile elements have a diameter in the range of l-100μm, preferably l-20μm.

29. A method according to any preceding claim, wherein the travel of the mobile elements through the chamber is monitored in real time by a microscopic method.

30. A method according to claim 29, wherein the travel of the mobile elements through the chamber is monitored in real time by fluorescence microscopy.

31. A method according to any preceding claim, wherein the chamber is maintained at a predetermined temperature.

32. A method according to any preceding claim, wherein the or at least one of the one or more further fluids or the mobile element receiving fluid has a composition which causes a species carried by the mobile elements to dissociate from the mobile elements.

33. A method according to any preceding claim, wherein mobile elements leaving the fluidic device are passed into a second fluidic device and moved across a plurality of fluid streams in the second fluidic device.

34. A fluidic device comprising: a chamber; at least three inlets leading into the chamber; at least one outlet arranged across the chamber from the inlets; at least three reservoirs in fluid communication with the inlets so that a respective fluid can be held in each reservoir and passed separately from the other fluids from the reservoir to the chamber via a corresponding at least one of the inlets; the shape and dimensions of the chamber being such that fluids passed from the reservoirs can be passed through the chamber in respective, parallel fluid streams to the at least one outlet wherein each fluid stream contacts at least another one of the fluid streams.

35. A fluidic device according to claim 34, wherein one or more of the reservoirs is in fluid communication with a plurality or a respective different plurality of the inlets.

36. A fluidic device according to claim 35, wherein the or each plurality of inlets is connected to the corresponding reservoir via a respective branched channel.

37. A fluidic device according to any one of claims 34 to 36, wherein the fluidic device includes a magnet positioned to apply a magnetic gradient across the fluid streams.

38. A fluidic device according to claim 37, wherein the magnet is a permanent magnet.

39. A fluidic device according to claim 38, wherein the magnet is an NdFeB magnet.

40. A fluidic device according to any one of claims 34 to 39, including a detector.

41. A fluidic device according to claim 40, wherein there are at least two said outlets, a first one of the outlets being positioned for receiving a fluid stream of the fluid from one of the reservoirs and for carrying said fluid to the detector.

42. A fluidic device according to any one of claims 34 to 41, wherein the chamber has a depth and a width, the depth being less than the width, and the fluid streams lying side-by- side across the width of the chamber.

43. A fluidic device according to claim 42, when dependent on claim 35 or 36, wherein the inlets of the or each plurality of inlets are spaced in a direction across the width of the chamber.

44. A fluidic device according to any one of claims 34 to 43, wherein at least one of the reservoirs comprises a syringe.

45. A fluidic device according to any one of claims 34 to 44, including a temperature control device for controlling the temperature of the chamber.

Description:

METHOD OF OPERATING A FLUIDIC DEVICE AND A FLUIDIC DEVICE FOR USE IN THE METHOD

The invention relates to a method of operating a fluidic device and also to a fluidic device for use in the method.

In accordance with a first aspect of the invention, there is provided a method of operating a fluidic device comprising: introducing a fluid carrying mobile elements, a mobile element receiving fluid and one or more further fluids into a chamber; passing the fluids through the chamber in respective, parallel fluid streams wherein each fluid stream contacts at least another one of the fluid streams; and applying a force or forces to the mobile elements to cause the mobile elements to move from the fluid stream of the mobile element carrying fluid through the fluid stream or sequentially through the respective fluid streams of the one or more further fluids and then into the fluid stream of the mobile element receiving fluid.

In accordance with a second aspect of the invention, there is provided a method of operating a fluidic device comprising: introducing a fluid carrying mobile elements, and one or more further fluids, into a chamber; passing the fluids through the chamber in respective, parallel fluid streams wherein each fluid stream contacts at least another one of the fluid streams; and applying a force or forces to the mobile elements to cause the mobile elements to move from the fluid stream of the mobile element carrying fluid and into the or sequentially into each fluid stream of the one or more further fluids; wherein each mobile element carries a

first species and the or at least one of the one or more further fluids carries a second species which binds to the first species.

In accordance with a third aspect of the invention, there is provided a fluidic device comprising: a chamber; at least three inlets leading into the chamber; at least one outlet arranged across the chamber from the inlets; at least three reservoirs in fluid communication with the inlets so that a respective fluid can be held in each reservoir and passed separately from the other fluids from the reservoir to the chamber via a corresponding at least one of the inlets; the shape and dimensions of the chamber being such that fluids passed from the reservoirs can be passed through the chamber in respective, parallel fluid streams to the at least one outlet wherein each fluid stream contacts at least another one of the fluid streams.

The following is a more detailed description of embodiments of the invention, by way of example, reference being made to the appended schematic drawings in which:

Figure 1 shows a first method being performed in a first micro-fluidic device;

Figure 2 shows a second micro-fluidic device;

Figure 3 shows a second method being performed in the second micro-fluidic device; and

Figure 4 shows a third micro-fluidic device.

Figure 1 shows the internal configuration of the first micro-fluidic device 10. The first micro-fluidic device 10 has an internal chamber 12. At one end of the internal chamber 12, a row of thirteen parallel inlet channels 14a, 14b, 14c, 14d, 14e open into the internal chamber 12 at, respectively, a row of thirteen inlets 16a, 16b, 16c, 16d, 16e. At the other

_

3 end of the internal chamber 12, a row of thirteen parallel outlet channels 18a, 18b, 18c, 18d, 18e communicate with the internal chamber 12 via, respectively, a row of thirteen outlets 20a, 20b, 20c, 2Od, 2Oe.

The length of the internal chamber 12 between the inlets 16a-16e and the outlets 20a-20e is about 5mm to 10mm (the drawing is not to scale). The width of the internal chamber 12, which is the dimension along which the inlets 16a-16e and the outlets 20a-20e are spaced, is about 2mm to 3 mm. The width of each inlet channel 14a-14e, each inlet 16a-16e, each outlet 20a-20e and each outlet channel 18a-18e is about 100 micrometers in the direction of the width of the internal chamber 12.

The first micro-fluidic device 10 may be formed in a conventional manner using a lower glass plate (not shown) and an upper glass plate (not shown). During the manufacture of the first micro-fluidic device 10, the internal chamber 12, the inlet channels 14a-14e and the outlet channels 18a-18e are etched into a planar surface of the lower glass plate. A planar surface of the upper glass plate is then heat-annealed to the lower glass plate so that the upper glass plate closes the internal chamber 12, the inlet channels 14a-14e and the outlet channels 18a-18e. The depth of etching is about 20 micrometers and so, after completion of the micro-fluidic device 10, the depth of the internal chamber 12, the depth of the inlet channels 14a-14e and the depth of the outlet channels 18a-18e is about 20 micrometer. The internal chamber 12 is formed between generally parallel, planar surfaces on the upper and lower glass plates, respectively.

The method of manufacture of the micro-fluidic device 10 is not important and the device 10 may be made using any suitable method.

Starting from the bottom of Figure 1, a first one of the inlet channels 14a leads to a first reservoir (not shown) which contains a first liquid. Second, third and fourth ones of the inlet channels 14b lead to a second reservoir (not shown) which contains a second liquid. Fifth, sixth and seventh ones of the inlet channels 14c lead to a third reservoir (not shown) which contains a third liquid. Eighth, ninth and tenth ones of the inlet channels 14d lead to fourth reservoir (not shown) which contains a fourth liquid. Eleventh, twelfth and thirteenth ones of the inlet channels 14e lead to a fifth reservoir (not shown) which contains a fifth liquid.

At the outlet end of the internal chamber 12, and again starting from the bottom of Figure 1, first to tenth ones of the outlet channels 18a, 18b, 18c, 18d lead to a common waste reservoir. The eleventh, twelfth and thirteenth outlet channels 18e lead to a detector (not shown) which may be of any suitable type but in this particular case is a fluorescence detector.

As shown in Figure 1, a strong permanent magnet 22 is fixed relative to the internal chamber 12 so as to apply a magnetic gradient across the width of the chamber 12. The magnet 22 is preferably an NdFeB magnet.

The operation of the first micro-fluidic device 10 will now be described generically, although it will be appreciated that an infinite number of methods made be performed with

W

5 the device 10 depending on the respective compositions of the first, second, third, fourth and fifth liquids.

In the exemplary, generic method now described, the first liquid in the first reservoir consists of a buffer and mobile elements in the form of micro-magnetic particles carried in suspension in the buffer. The magnetic particles are shown at 24 in Figure 1. The particles 24 have a superparamagnetic core which is coated by an external, generally inert polymeric shell. The particles have a diameter of about 2.8 micrometers. Particles of this type are available commercially, for example, under the trade mark DYNABEADS (TM). The surface of the magnetic particles 24 is functionalized with a reactive group such as carboxy or amino, and a first species (which may, for example, be a molecule or a cell as discussed in more detail below) is bound to the reactive group. The first species is shown at 26 in Figure 1.

The second liquid, contained in the second reservoir, consists of the same buffer used for the first liquid (without the particles 24) and a second species which may be, for example, a molecule or a cell and which is carried in supension or solution in the buffer. The second species is shown at 28 in Figure 1. The second species 28 binds specifically to the first species 26.

The fourth liquid in the fourth reservoir consists of the same buffer used in the first and second liquids and a third species 30 which may also be, for example, a molecule or a cell and which is carried in solution or suspension in the buffer. The third species 30 binds

6 specifically to the second species 28, (or to the second species 28 and the first species 26 when the second species 28 is bound to the first species 26). The third species 30 may be fluorescent, or may have some other property which facilitates detection as discussed below.

Finally, the third and fifth liquids in the third and fifth reservoirs consist of buffer only and are identical to one another.

The first, second, third, fourth and fifth liquids are then pumped, simultaneously, from their respective reservoirs, through the corresponding inlet channels 14a-14e, through the internal chamber 12 and into the outlet channels 18a-18e. Pumping may be achieved by any suitable method such as, for example, using commercially available syringe pumps. When syringe pumps are used, each reservoir may be the liquid holding space of a respective syringe.

As seen in Figure 1, the first liquid passes through the first inlet channel 14a, through the corresponding inlet 16a, across the internal chamber 12, through the outlet 20a, and into and through the outlet channel 18a. The second liquid passes through the second, third and fourth inlet channels 14b, through the corresponding second, third and fourth inlets 16b, through the internal chamber 12, through the second, third and fourth outlets 20b and finally through the second, third and fourth outlet channels 18b. The third liquid passes through the fifth, sixth and seventh inlet channels 14c, through the corresponding fifth, sixth and seventh inlets 16c, through the internal chamber 12, through the fifth, sixth and seventh outlets 20c, and finally through the fifth, sixth and seventh outlet channels 18c. The fourth liquid passes through the eighth, ninth and tenth inlet channels 14d, through the corresponding eighth,

.

7 ninth and tenth inlets 16d, through the internal chamber 12, through the eighth, ninth and tenth outlets 2Od, and finally through the eighth, ninth and tenth outlet channels 18d. Finally, the fifth liquid passes through the eleventh, twelfth and thirteenth inlet channels 14e, through the corresponding eleventh, twelfth and thirteenth inlets 16e, through the internal chamber 12, through the eleventh, twelfth and thirteenth outlets 2Oe, and finally through the eleventh, twelfth and thirteenth outlet channels 18e.

As shown in Figure 1, each liquid passes across the internal chamber 12 in a respective, generally distinct liquid stream. The first liquid passes across the chamber 12 in a first liquid stream 32, the second liquid passes across the chamber 12 in a second liquid stream 34, the third liquid passes across the chamber 12 in a third liquid stream 36, the fourth liquid passes across the chamber 12 in a fourth liquid stream 38, and the fifth liquid passes across the chamber 12 in a fifth liquid stream 40. The first, second, third, fourth and fifth liquid streams 32 to 40 lie parallel to one another side-by-side across the width of the internal chamber 12. There are no structural divisions within the internal chamber 12 and, as seen in Figure 1, the first liquid stream 32 contacts the second liquid stream 34 which also contacts the third liquid stream 36. The third liquid stream also contacts the fourth liquid stream 38, and the fourth liquid stream 38 also contacts the fifth liquid stream 40. Due to the small height dimension of the internal chamber 12, the liquid streams 32 to 40 pass through the chamber 12 under conditions of laminar flow. This means that turbulent mixing between contacting liquid streams is substantially avoided. Some diffiisional mixing between contacting liquid streams does occur. However, the fluid flow rate, the respective widths of the liquid streams, and the length of the internal chamber 12 are such that the first to fifth

liquid streams 32 to 40 remain generally distinct throughout the length of the internal chamber 12. The width of the first liquid stream 32 is about 200 micrometers. The width of each of the second, third, fourth and fifth liquid streams 34 to 40 is about 700 micrometers.

As the magnetic particles 24 enter into the internal chamber 12, they come under the influence of the magnetic gradient generated by the permanent magnet 22. This causes the superparamagnetic cores of the magnetic particles 24 to become temporarily magnetized and so a magnetic attraction arises between each individual magnetic particle 24 and the permanent magnet 22. As shown in Figure 1, this magnetic attraction draws the individual magnetic particles 24 out of the first liquid stream 32 and sequentially into and through each of the second, third and fourth liquid streams 34, 36, 38. Finally, the individual magnetic particles 24 are pulled into the fifth liquid stream 40 where they exit the internal chamber 12 through any one of the eleven, twelfth or thirteenth outlets 2Oe and pass into any one of the eleventh, twelfth or thirteenth outlet channels 18e.

As the magnetic particles 24 pass through the second liquid stream 34, the first species 26, which is bound to the magnetic particles 24, binds to the second species 28. The magnetic particles 24, which are now carrying both the first species 26 and the second species 28, then pass into the third liquid stream 36 which consists of the third liquid, which in this example is buffer alone. The magnetic particles 24, still carrying both the first and second species 26, 28, are drawn across the third liquid stream 36 by the magnetic attraction, into the fourth liquid stream 38. It is possible that small amounts of unbound second species 28 will pass into the third liquid stream 36. However, without any magnetic attraction acting on the

^

9 unbound second species 28, the unbound second species 28 is unable to cross the third liquid stream 36 and will, instead, exit the internal chamber 12 via one of the third stream outlets 20c. On entry of the magnetic particles 24 into the fourth liquid stream 38, the second species 28, which is bound via the first species 26 to the magnetic particles 24, becomes bound to the third species 30. Finally, the magnetic particles 24, now carrying the first species 26, the second species 28 and the third species 30 pass into the fifth liquid stream 40 and then exit the internal chamber 12 via the eleventh, twelfth and thirteenth outlets 2Oe and via the eleventh, twelfth and thirteen outlet channels 18e for detection at the detector (not shown). In this specific example, the detector is a fluorescence detector and this detects the fluorescent third species 30.

If desired, an extra liquid stream (not shown) may be incorporated between the fourth and fifth liquid streams 38, 40. This would be used to wash away any unbound third species 30 and prevent any such unbound third species 30 from passing into the fifth liquid stream 40. Alternatively, the use of such an additional "wash" liquid stream may be avoided if the detector is connected only to the final, thirteenth outlet channel 18e. In this case the magnetic particles 24, now carrying the first 26, second 28 and third species 30 pass into the thirteenth outlet channel 18e to the detector and any unbound third species 30 passes into either the eleventh or twelfth outlet channel 18e.

Although the operation of the first micro-fluidic device 10 has been described in generic terms, it will be appreciated that the device 10 may be used to conduct an infinite number of different methods. For example, the first micro-fluidic device 10 shown in Figure 1 is

particularly suited for carrying out molecular recognition type methods or chemical/biochemical synthesis type methods.

For example, the device 10 may be used in a molecular recognition procedure in which the second liquid is a sample having an unknown content of a particular species, such as a molecule or a cell. By way of a more specific example, the first species 26 bound to the magnetic particles 24 in the first liquid may be a specific antigen. The second liquid may be a sample having an unknown content of a particular antibody specific for that antigen - the antibody corresponding to the second species 28. The third species 30 may be a marker molecule such as, for example, a fluorescently labeled IgG specific antibody. As the first liquid enters into the internal chamber 12, the magnetic particles 24 carrying the antigen (corresponding to the first species 26) pass into the second liquid stream 34 whereupon the antigen 26 binds to the target antibody (corresponding to the second species 28). As the magnetic particles 24 carrying the antigen 26 and the target antibody 28 pass into the fourth liquid stream 38, the fluorescently labeled IgG specific antibody (corresponding to the third species 30) binds onto the target antibody 28. The magnetic particles 24, now carrying the antigen 26, the target antibody 28 and the fluorescently labeled IgG specific antibody 30 now pass into the fifth liquid stream 40 and to the detector where suitable detection methodology allows detection of and quantification of the target antibody 28 in the second sample liquid.

A second exemplary molecular recognition method may detect and quantify a specific antigen contained in a sample liquid (corresponding to the second liquid). In this case, the

first species 26 is an antibody specific for the antigen, the antigen corresponds to the second species 28, and the third species 30 corresponds to a marker molecule which also binds to the antigen 28.

A third exemplary molecular recognition type procedure is intended to detect and isolate a desired single stranded nucleic acid sequence. In this example, the first species 26 is a single stranded nucleic acid sequence which is complimentary, or partially complimentary, to the single stranded nucleic acid sequence to be isolated. The sequence to be isolated is the second species 28 contained in the second liquid. In this case, there is no need for a third species 30. However, a third species 30 may be used for the purposes of quantifying the isolated nucleic acid sequence. For example, the third species 30 may be a fluorescent marker which binds only to double stranded nucleic acid - and so which will bind to the first and second species 26, 28 once they have annealed together.

Alternatively, the first micro-fluidic device 10 may be used for performing chemical or biochemical synthesis. In this case, the first, second and third species 26, 28, 30 are, for example, molecular species which bind together, for example by covalent bonding. For example, the device 10 may be used to form nucleotides with the first, second and third species 26, 28, 30 being individual nucleotide bases which become covalently linked together in a specific order.

In the generic example discussed above, it will be noted that each of the second, third, fourth and fifth liquid streams 34, 36, 38, 40 is fed, respectively, by three corresponding inlet

channels 14b, 14c, 14d, 14e and three corresponding inlets 16b, 16c, 16d, 16e. The use of a plurality of inlets to feed a single liquid stream is advantageous because it helps to achieve homogeneity of fluid flow rate at different positions in a cross-section taken perpendicular to the direction of liquid flow. It also helps to avoid sudden pressure changes which might tend to disrupt laminar flow. However, the use of a plurality of inlets for each liquid stream is not essential and if a single inlet is used for a single liquid stream, there will tend to be a greater flow rate at the center of the stream as compared to the outer edges of the stream. Because a liquid stream will tend to have a greater dimension across the width of the internal chamber 12, as compared to the height of the liquid stream (which corresponds to the height of the internal chamber 12), when a plurality of inlets are used to feed a single liquid stream, it is desirable to space the inlets from one another across the width of the stream.

A second micro-fluidic device 42 is shown in Figure 2. The second device has an internal chamber 44 which is about 6mm long, about 3mm wide, and about 20 micrometers deep.

At one end of the internal chamber 44, the chamber 44 is in fluid communication with four inlet channels 46, 48, 52, 56. Starting from the bottom of Figure 2, a first inlet channel 46 opens into the internal chamber 44 at a single inlet 47. A second inlet channel 48 divides so as to open into the internal chamber 44 at eight adjacent inlets 50. A third inlet channel 52 divides so as to open into the internal chamber 44 at eight adjacent inlets 54. Finally, a fourth inlet channel 56 divides so as to open into the internal chamber 44 at eight adjacent inlets 58.

The other end of the internal chamber 44 is provided with 32 outlets 60 which are spaced across the width of the chamber 44. The 32 outlets 60 feed a network 62 of branched channels which, in turn, lead into a common drain 64.

The first inlet channel 46 communicates with a first reservoir 66, the second inlet channel 48 communicates with a second reservoir 68, the third inlet channel 52 communicates with a third reservoir 70 and the fourth inlet channel 56 communicates with a fourth reservoir 72. The first, second, third and fourth reservoirs 66, 68, 70, 72 may be, for example, the fluid spaces of respective syringe pumps.

As for the first micro-fϊuidic device 10, the second micro-fluidic device 42 may be manufactured by etching the chamber 44 and the inlet and outlet channels 46, 48, 52, 56, 62, 64 into a lower glass plate (not shown) and then by annealing an upper glass plate (not shown) so as to close the chamber and channels. A plurality of posts (shown as seven white dots in chamber 44) are spaced throughout the internal chamber 44. Each post is an area of the lower glass plate which has not been etched and thus forms a post which extends the full height of the internal chamber 44. The posts serve as spacers helping to prevent inadvertent collapse of the chamber 44 during heat annealing of the upper and lower glass plates. In practice, it has been found that the posts do not adversely affect laminar flow of fluid streams through the internal chamber 44 - the fluid streams simply flowing around the posts. (The posts may be dispensed with when the upper and lower plates are not connected by heat annealing or when there is no tendency for the chamber to collapse during manufacture.)

As seen in Figure 3, a permanent magnet 76 is positioned adjacent to the internal chamber 44 so as to apply a magnetic gradient across the length of the chamber 44.

In the second micro-fluidic device 42, the outlets 60 do not lead to a detector. Instead, the second micro-fluidic device 42 is used with a fluorescence microscope which is arranged so that the whole of the internal chamber 44 can be viewed simultaneously in real time (the micro-fluidic device 42 being made from transparent glass plates). The second micro-fluidic device 42 has been used to perform a method intended to demonstrate that real time fluorescence microscopy can be used to track the passage of individual magnetic particles across the internal chamber 44. Three sequential views taken in real time with a fluorescence microscope from this experiment are shown in Figure 3.

In order to conduct the experiment, a first liquid consisting of buffer carrying micro magnetic particles was placed in the first reservoir 66. In this case, streptavidin had been bound to the surface of the particles. A second liquid, consisting of buffer alone, was placed in the second reservoir 68. A third liquid, consisting of buffer and biotin-FITC in solution in the buffer was placed into the third reservoir 70. Finally, a fourth liquid consisting of buffer alone was placed into the fourth reservoir 72. The four liquids were then pumped simultaneously through the respective inlet channels 46, 48, 52, 56, through the respective inlets 47, 50, 54, 58 into the internal chamber 44 and from the internal chamber 44 via the outlets 60, through the network of branched channels 62 and into the common drain 64. This resulted in the passage of four, generally distinct, parallel liquid streams along the internal chamber 44. A first, relatively narrow liquid stream was made up of first liquid

.

15

(buffer carrying micro magnetic particles) and passed along the internal chamber 44 from the single inlet 47. A second liquid stream consisted of the second liquid (buffer alone) and passed along the internal chamber 44 from the corresponding group of eight inlets 50. A third liquid stream consisted of the third liquid (buffer carrying biotin-FITC) and passed along the internal chamber 44 from the corresponding group of eight inlets 54. Finally, a fourth liquid stream consisting of the fourth liquid (buffer alone) passed along the internal chamber 44 from the corresponding group of eight inlets 58. In Figure 3, the first and second liquid streams are shown together at 78, the third liquid stream is shown at 80 and the fourth liquid stream is shown at 82.

During the passage of the liquid streams along the internal chamber 44, the micro-magnetic particles became temporarily magnetized and were attracted towards the magnet 76. This caused the micro-magnetic particles to be pulled from the first liquid stream sequentially through the second and third 80 liquid streams to the fourth liquid stream 82. The passage of individual particles 81 across the internal chamber 44 was observed in real time using the fluorescence microscope. No fluorescence was observed until the particles 81 began to approach the third liquid stream 80 (the third liquid stream contains biotin-FITC). In Figure 3, fluorescent particles 81 are shown circled, with (in some cases) an arrow to show direction of travel. As shown in Figure 3a, individual particles start to become fluorescent as they approach the third liquid stream 80. As the particles enter into the third liquid stream 80, reaction between the streptavidin coated particles and the biotin-FITC reaches completion and all of the particles fluoresce. This is shown in Figure 3 b. Finally, as the particles 81 which now carry both streptavidin and biotin-FITC enter into the fourth liquid

stream 82 (consisting of buffer alone), the particles remain fluorescent indicating the binding of the streptavidin with the biotin-FITC.

Figure 4 shows a third micro-fluidic device 90. The third micro-fluidic device 90 is identical to the first micro-fluidic device 10 with the exception that the chamber 92 is trapezoidal in shape rather than rectangular as in the case of the chamber 12. The third micro-fluidic device 90 is included to demonstrate that the shape of the chamber may vary, and will not be described in detail.

In the examples described above, the mobile elements are in the form of particles and more specifically micro-magnetic particles. However, other forms of mobile elements may be used. A mobile element can be any element which can be carried by the fluids (for example in suspension or in solution in the fluids) and which can be moved through the fluids by the applied force or forces. The mobile elements may be in the form of particles, such as synthetic particles. The mobile elements may also be cells. One possibility is to use cells with nanomagnetic particles attached thereto. Alternatively, the mobile elements may take the form of droplets, such as a water in oil droplet. Other examples of suitable mobile elements are polymeric particles and large biomolecules. When the force used to move the mobile elements is not a magnetic force, then the magnetic characteristics of the mobile elements are not important. When particles are used, ideally they are in the micrometer size range, for example 1 micrometer to 10 micrometers, but larger and smaller particles may be used.

When the mobile elements are non-permeable particles, species may be bound to the surface of the particles. When the mobile elements are droplets, porous particles, micelles or liposomes, for example, species may be carried, and react with other species, either on the surface or within the mobile elements.

In the examples described above, magnetic attraction between a permanent magnet and superparamagnetic particles is used to draw the particles across the fluid streams. However, any suitable force may be used to move the mobile elements across the fluid streams. For example, if the mobile elements take the form of diamagnetic particles, a strong magnet, such as a superconducting magnet, may be used to induce diamagnetic repulsion so as to move the particles across the fluid streams. Diamagnetic repulsion may also be achieved using conventional magnets with careful design of the magnetic field. Repulsion forces may be enhanced by implementation of diamagnetic features in the vicinity of the chamber. Instead of magnetism, gravity may be used to move the mobile elements across the fluid streams. For example, if the mobile elements are denser than the fluids through which the elements are to be drawn, gravity will tend to make the elements move downwards. The fluid streams will then be arranged vertically, one above another. Alternatively, by using mobile elements which are less dense than the fluids, gravity may be used to cause the elements to rise through fluid streams due to buoyancy. The use of gravity as a force to move the mobile elements across the fluid streams is less desirable than magnetic attraction, because it is possible to achieve greater forces using magnetic attraction. In cases where gravity is insufficient, centrifugal forces may be used to move the mobile elements across the fluid streams. Other forces which may be used to move the mobile elements across the

fluid streams may be generated by dielectrophoresis, ultrasonic forces or by using optical tweezers. Different forces may be used in combination.

When magnetic attraction is to be used as a force, a NdFeB magnet is a prefered type of magnet for generating a magnetic field, because it is relatively inexpensive and produces a relatively strong magnetic field. However, where a magnetic force is to be used, any suitable method of generating a magnetic field may be used. For example, a magnetic field can be generated with a SmCo permanent magnet, an electromagnet, a miniaturised electromagnet such as a MEMS device, or a hybrid consisting of a small magnetisable feature or features in the vicinity of the chamber that is/are magnetically connected to a larger external magnet.

An electromagnet may be used to hold mobile elements immobile in a desired position in the chamber - the elements being released once the electromagnet is de-energised.

In the examples given above, the particles 24, 81 move in a single direction across the fluid streams. However, it is possible to make the mobile elements move in a zig-zag or sawtooth motion back and forth across the fluid streams by using, for example, a plurality of magnets spaced along the length of the internal chamber 12, 44 on opposite sides of the internal chamber.

Any suitable fluids may be used to form the fluid streams. Examples of suitable fluids are water, aqueous solutions, non aqueous solutions, non-aqueous solvents, ionic liquids and biological fluids. Contacting fluid streams are always different from one another in some

respect. Preferably, they will be different from one another in composition. Alternatively they may differ from one another in temperature. Although fluids which are identical to one another in all respects can be fed into the chamber via adjacent inlets, they are considered to form a single fluid stream as there is no discernable interface. Fluid streams which do not contact one another may be identical or different from one another. When contacting fluid streams differ from one another in composition, the compositions may differ from one another in any way. For example, the different fluids may be based on the same liquid, such as an aqueous buffer, and differ in terms of the presence/absence, nature or concentration of a species carried either in solution or suspension in the liquid. Alternatively, the respective fluids of contacting fluid streams may differ from one another in more fundamental ways. For example, they may comprise different aqueous buffers. It is also possible for one fluid stream to be aqueous in nature and for a contacting fluid stream to be non-aqueous in nature. It is also possible for the respective fluids of contacting fluid streams to be immiscible - such as an aqueous liquid and an organic solvent. In such cases, known methods may be employed to avoid the formation of an emulsion at the interface between the immiscible liquids. For example, the chamber surfaces bordering an aqueous fluid stream may be given a hydrophilic surface coating and the chamber surfaces bordering a contacting hydrophobic flow stream may be given a hydrophobic surface coating. Alternatively, the chamber surfaces bordering the chamber may be shaped so as to help to avoid the formation of an emulsion at the interface between an aqueous fluid stream and a fluid stream comprising a water-immiscible organic solvent.

It is also possible to choose a fluid stream so as to cause dissociation of species bound (directly or indirectly) to mobile elements. For example, dissociation may be caused by choosing a fluid stream having an appropriate ionic strength or by choosing a fluid stream comprising an organic solvent.

The dimensions of the internal chambers may be varied from those of the examples given above. However, in general, the dimensions will be chosen so as to ensure laminar flow of the fluid streams. In general, this will involve giving the internal chamber a relatively small dimension in one direction (the height in the examples given above). This limiting dimension will generally be less than lmm as laminar flow starts to breakdown as the limiting dimension approaches lmm. Preferably, the limiting dimension will be in the range of 1 micrometer to 500 micrometers. More preferably, the limiting dimension will be in the range of 1 micrometer to 100 micrometers.

The length of the internal chamber, that is to say the dimension between the inlets and the outlets, is generally kept as short as possible. This is because, for a given flow rate, the longer the chamber the greater the residence time of the fluid in the chamber. It is desired to minimise the length because greater residence times lead to greater degrees of mixing by diffusion.

The width of the chamber may be increased where it is desired to increase the number of fluid streams. It may be possible to obtain 30 or more fluid streams lying side-by-side across the width of the chamber. This will allow a greater number of steps to be performed

in multi-step methods such as molecular recognition methods or chemical/biochemical synthetic methods. For chambers with greater widths, it is desirable to use particularly strong magnets, so as to apply greater forces to the particles, this is because the particles will generally have a greater lateral distance to travel across the fluid streams and so greater forces are desired in order to minimise the length of the chamber. Alternatively, a series of integrated microfabricated magnets may be used to pull the mobile elements from one stream to a neighbouring stream, to hold them within a stream, and to release them to the next stream, as desired.

As shown in the embodiment described above in relation to Figure 2, it is not necessary to have a respective outlet corresponding to each inlet. It is possible to use unequal numbers of inlets and outlets. A single common outlet could be used for all fluid streams.

In the examples given above, the fluid streams are arranged side-by-side in a single direction (the width of the chambers described above). However, this need not be the case and fluid streams may be arranged such that some fluid streams lie side-by-side in a first direction and other fluid streams lie side-by-side in a second direction arranged, for example, perpendicularly to the first direction. For example, the fluid streams may be arranged in two, generally planar, parallel layers, each layer consisting of a number of side-by-side fluid streams and the fluid streams of one layer contacting the fluid streams of the adjacent layer.

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Conveniently, the fluid streams extend in straight lines through the chamber. However, this need not be the case. Fluid streams may flow in parallel curves through a suitably shaped chamber.

It is also possible for fluid streams to flow in opposite and parallel directions. This could be used, together with suitably placed magnets, to cause the mobile elements to travel around a circular path, for one or more revolutions, until they are released into an outlet channel.

The first and second micro-fluidic devices 10, 42 described above are each manufactured from two glass plates by etching and annealing. However, fluidic devices in accordance with the invention may be made by any suitable method. Fluidic devices may be formed from materials other than glass, such as: quartz; silicon; polymers such as PMMA, PC, PDMS and COC; ceramic materials; and metals such as stainless steel and aluminium.

Any suitable method of pumping the fluids through the chamber may be used. For example, any method of generating hydrostatic pressure, such as the application of positive or negative pressures may be used. Electroosmotic or shear driven flow may also be used.

The methods of the invention may or may not include detection. In the examples given above, detection is achieved using fluorescence - although other methods of detection may be used. Other methods suitable for detection include: phosphorescence; chemiluminescence; bioluminescence; spectroscopic methods such as Raman or SERRS;

..

23 magnetic detection; radioisotope detection; optical detection; electrochemical detection; and mass spectrometry.

There are a large number of parameters which affect reactions carried out in accordance with the current invention. These include: fluid flow speeds, widths of the flow streams, strength of the applied force (e.g. magnetic force), susceptibility of the mobile elements to the force, diameter of the mobile elements, concentration of reacting species in the fluids, number of functionalized groups bound to the mobile elements and the temperature. All of these parameters may be optimized for a particular desired method. These parameters should be adjusted so that the residence time of the mobile elements in any particular fluid stream is sufficient for the binding/reaction which is to occur in that fluid stream. In order to achieve this, use may be made of the following equations.

The mobile element's flow path through the micro-fluidic chamber can be described as a velocity vector u partic i e . At the given position in the chamber, u partic i e is the sum of the vector U flow for the applied pumping rate and u mag , the magnetically induced velocity vector towards the surface of the magnet (equation 1):

U pa r t i c l e

The vector u flow is aligned along the x-direction (see Figure 1), the orientation of the magnetically induced flow vector u mag will depend on the magnetic field design, but should be primarily oriented in the y-direction. It can be shown that the magnetically induced velocity is proportional to the saturation magnetization of the mobile element M s (in A m 2 )

and the gradient of the applied magnetic field gradH (in Tm "1 ), and that u mag is inversely proportional to the viscosity of the medium η (in kg m '1 s "1 ) and the radius of the mobile element r (in m) (equation 2).

It may be desirable to use a temperature control device, such as a thermostatic heater, so as to control the temperature of the chamber. For example, the temperature of the chamber may be kept constant. Viscosity of the fluid streams is greatly affected by temperature and the use of a temperature control device may help to maintain viscosities of the fluid streams at acceptable values. Alternatively, it may be desirable to change the chamber temperature in a predetermined manner so as to study reaction kinetics.

In the examples given above, a single species is bound to the mobile elements when the mobile elements are introduced into the chamber. However, this need not be the case. It is possible to use mobile elements with several functionalities that upon binding would give different signals (for example different colours of fluorescence). It is also possible to use different types of mobile elements which are "bar-coded" so that each bar-code identifies a different respective type of mobile element having a different surface coating (for example different species bound to different respective mobile elements).

The current invention is applicable to a number of different types of methods. For example, it is applicable to molecular detection type procedures where species such as molecules, cells or intra-cellular components are detected or both detected and quantified. By using a larger number of fluid streams, it is possible to use a greater number of species so as to allow

detection of two or more species simultaneously. The methods are also applicable to chemical or biochemical synthetic reactions in which a plurality of different steps are carried out sequentially in different fluid streams. Alternatively, the current methods are applicable to multi-step molecular modification reactions in which a species bound to a mobile element is subjected to successive chemical modifications as it passes through successive fluid streams. It is also possible to use the current methods to analyse hydrophobicity/hydrophilicity of mobile elements and liquids. For example, a mobile element with a hydrophobic surface will require more force than a hydrophilic mobile element to be pulled from an organic solvent stream into an aqueous solvent stream. If set up carefully the surface tension at the mobile element-liquid interface may be measured by determining the level of magnetic force required to pull the mobile element through a liquid- liquid interface.

It is also possible to use a cascade of two or more fluidic devices. In this case, the mobile elements leaving one fluidic device would be fed into another fluidic device, where they may then be passed through a plurality of fluid streams. In this way, the number of reaction steps or recognition steps, for example, may be increased many fold.

The methods and devices described above benefit from a number of advantages. Firstly, the methods are often capable of being performed on a continuous basis, rather than on a batch basis. Secondly, the methods can be performed with very small quantities of fluids containing small quantities of the various species/reagents.