WO2009068026A1

WO2009068026A1 - Micro capillaries and a separation device comprising them

Info

Publication number: WO2009068026A1
Application number: PCT/DK2007/000518
Authority: WO
Inventors: Peter Warthoe; Per BERDÉN
Original assignee: Atonomics A/S
Priority date: 2007-11-26
Filing date: 2007-11-26
Publication date: 2009-06-04

Abstract

The present invention relates to a capillary channel comprising one or more capillary microchannels enhancing the capillary drag force of the capillary channel. The present invention relates to a device for separating a suspension into a liquid phase and a retentate phase. The device comprises a separation chamber comprising an application zone and a hydrophilic filter material. The separation chamber is connected to a first capillary channel, where the connecting junction between the separation chamber and the first capillary channel comprise a capillary micro channels and optional a physical barrier preventing flow of residue retentate from a lower part of the chamber into the first capillary channel. The invention further relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter.

Description

Title: Micro capillaries and a separation device comprising them

Technical Field

The present invention relates to a capillary channel comprising one or more capillary microchanneis enhancing the capillary drag force of the capillary channel.

The invention further relates to a device for separating a suspension into a liquid phase and a retentate phase comprising the capillary channels according to the invention.

The invention further relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter. The suspension might be blood, the liquid phase plasma/serum and the retentate blood cells.

Background

Many diagnostics are carried out in the clinical field utilizing blood as a sample. Although some of these techniques can be carried out on whole blood, it is necessary in many instances to utilize serum or plasma as the sample in order to obtain an accurate reading. For example, red blood cells (erythrocytes) scatter and absorb light and could adversely affect a measurement of either reflected or transmitted light of a diagnostic test relying on either of these measurement techniques.

Traditionally, plasma and serum have been separated from whole blood by centrifuging either before (for plasma) or after (for serum) clotting. However, centrifugation is time consuming and requires equipment that is not generally available outside the clinical laboratory. Accordingly, field testing of numerous blood substances that require serum or plasma is difficult.

A number of techniques have been devised to avoid this problem. The techniques generally utilize a filtering device capable of separating red blood cells from plasma. Numerous materials have been used in the past to form filters. Paper, non-woven fabric, sheet-like filter materia! composed of powders or fibres such as man-made fibres or glass fibres, and membrane filters having suitable pore sizes have been proposed. However, these prior art techniques have proven to be unsuitable for use in applica- tions which, because of space and volume restraints, can only utilize a small filter in a device in which a single drop of blood is separated and the plasma is transported through the device solely by means of capillary action. Thus, most prior art devices for separation suffers from dealing with sufficient to separate undiluted whole-blood by use of capillary and/or hydrostatic pressure without the use of an external force. Accordingly, further refinement in blood separation techniques is desirable.

Accordingly one object of the present invention was to develop a device and a method capable to separate undiluted whole-blood into a plasma/serum phase and a blood celt phase in a short time, where the plasma/serum phase is substantially free of blood ceil contamination.

An other object of the invention was to develop a device and a method capable to separate undiluted whole-blood into a plasma/serum phase and a blood cell phase in short time, where the separation is driven without the use of an external force.

Disclosure of the Invention

An object of the present invention was to develop an improved capillary channel as well as an improved device for separating a small liquid suspension into a liquid phase and a solid phase, without use of external force. Thus, an object was to develop a device comprising capillary channels wherein the internal drag force in the capillary channels was improved..

Surprisingly, the present inventors found that by creating microchannels in the capillary channels, these were much more efficient in providing the required drag force on the liquid sample.

Accordingly in one aspect, the invention relates to a capillary channel having a width of 0.25-2.0 mm and height of 0.2-1.0 mm, characterised in that the capillary channel comprise one or more capillary microchannels.

Accordingly, In one aspect the invention relates to a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device cσm- prising an application chamber (1) comprising a hydrophiiic filter material (17), said application chamber being connected to a capillary channel (3) according to the invention. In a further aspect the capillary channel comprises two or more capillary microchan- nels.

In a further aspect the capillary channel comprises three or more capillary microchan- nels.

An object of the invention was to develop a device and a method capable to separate a suspension into a liquid phase and a retentate phase in a short time, where the liquid phase is substantially free of retentate contamination, and where the separation is driven without the use of an external force.

In a further aspect of the invention the connecting junction between the application chamber and the capillary channel comprise a physical barrier (10, 10') preventing flow of residue retentate from a lower part of the chamber into the capillary channel. The physical barrier might be vertical or describes an incline extending from the bottom of the application chamber. The presence of this physical barrier was surprisingly shown to create a substantially improved separation of the fluid materia! from the suspended matter.

In a further aspect the device comprises an upper part and a lower part, where the two parts when assembled form an application chamber (1 ), a capillary channel (3), where the lower part of the capillary channel has one or more capillary micro channel(s) (11) enhancing the capillary drag force of the capillary channel, and said upper part having an inlet leading to the application chamber.

In a further aspect the interfaces between the upper and lower parts are sealed with a hydrophobic sealant.

In a further aspect the device further comprises a prefilter material ( 15).

In a further aspect the invention relates to the use of one or more capillary channels comprising on ore more micro channels for enhancing the flow of liquid into the capillary channel, In a further aspect the invention relates to the use of the device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, where the liquid phase is substantially free of suspended matter. The suspension might be blood.,

In a further aspect the invention relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of;

a. optionally applying a suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μi of a sample suspension, or the liquid of step a., to a filter material; c, applying the filter material comprising the suspension to an application chamber, which is connected to a capillary channel as defined above; d. over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel.

In a further aspect the invention relates to a method, where the liquid phase is directed into the capillary channel solely by the combined action of capillary forces provided by the capillary channel as defined above and by hydrostatic pressure generated by the applied sample. The blood might be human blood.

Brief Description of the Drawings

The invention is explained in detail below with reference to the drawings, in which

Fig, 1 illustrates a schematic presentation of a sample device comprising a microfluid channel having three chambers (3, 5, 6), an application zone (1), a separation chamber (2), a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a washing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10 (vertical), 10' (incline)) between the separation chamber and the first capillary channel, capillary micro channels (11 ) in the first capillary channel (3), corona treat- ment (12) (symbolised by the grey shade) of the first capillary channel, and a detector unit (14).

Fig. 2 illustrates same principle as in Fig, 1 with a three dimension illustration.

A sample device comprising a microfluid channel having three chambers (3, 5, 6), an application well (V), a separation chamber (2), a hydrophilic filter material (17) for blood filtration, a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a washing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10, 10') between the separation chamber and the first capillary channel (3), capillary micro channels (1 1) in the first capillary channel (3), corona treatment (12) of the first capillary channel (3) and a detector unit (14).

Fig. 3 illustrates a schematic site view of a separation device comprising a microfluid channel (3), an application well [V), a separation chamber (2), a first capillary channel (3), a physical barrier (10¹) between the separation chamber and the first capillary channel, a hydrophilic filter material (17), and a prefllter (15)

Fig, 4 illustrates a prototype picture of Fig. 2 presentation of a separation device comprising a microfluid channel having three chambers (3, 5, 6), a application well (1'), a separation chamber (2), a first capillary channel (3), a washing chamber (5), a detection chamber (6), a physical barrier (10') between the separation chamber and the first capillary channel, and a hydrophilic filter (17).

Fig. 5 illustrates a prototype picture of Fig. 4 (backside), presentation of an integrated separation and detection device comprising a microfluid channel having three chambers (3, 5, 6), an application well (V) backside, a separation chamber (2) backside, a first capillary channel (3), a washing chamber (5), a detection chamber (6), a physical barrier (10') between the separation chamber and the first capillary channel, and a hydrophilic filter (17). Left circle is a magnified view of the physical barrier (10') between the separation chamber and the first capillary channel in order to illustrate the capillary microchanneis (11) in the first capillary channel. Right circle is a magnified view of the first capillary channel at the collection chamber in order to illustrate the capillary micro- channels. Fig. 6 illustrates same principle as in Fig. 1 with a three dimension illustration including more features, A integrated separation and detection device comprising a microfluid channel having three chambers (3, 5, 6), an application well (V), a separation chamber (2), a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a wash- ing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10, 10') between the separation chamber and the first capillary channel, capillary micro channels (11) in the first capillary channel (3), a detector unit (14), a first compartment for detection solution A (9), a second compartment for detection solution B (15), a washing solution compartment (16), and a blood lid (12a).

Fig. 7A illustrates a schematic site view of an integrated separation and detection device comprising a microfluid channel (3,5,6), an application well (1'), a separation chamber (2) and the hydrophilic filter (17), a first capillary channel (3), serum/plasma (18) in the first capillary channel, signal solution (19) in washing (5) and detector chamber (6), light trap version A (20) in connecting junction between the first capillary channel (3) and the washing chamber (5), and a detector unit (14).

Fig. 7B illustrates a schematic site view of an integrated separation and detection de- vice comprising a microfluid channel (3,5,6), an application well (1¹), a separation chamber (2) and hydrophilic filter (17), a first capillary channel (3), serum/plasma (18) in the first capillary channel, signal solution (19) in washing (5) and detector chamber (6), a light trap version B (20') in connecting junction between the first capillary channel (3) and the washing chamber (5), and a detector unit (14),

Definitions

In the context of the present invention, by "capillary channel" is meant a narrow tube or channel through which a fluid can pass. Preferably the diameter (or with) of a capillary channel according to the invention is less than 10 mm. Even more preferred the diameter of a capillary channel according to the invention is less than 5mm, such as less than 4 mm, or less than 3 mm or even less than 2 mm. In a most preferred aspect the capillary channel has a diameter of 1 mm or less, e.g 0,2-1.0 mm. The channels may also be formed of non-circular shapes, e g, rectangular or triangular, in which case the "diameter" refers to the mean distance from the center of the cannel to the periphery, The terms "capillary channel" and "first capillary channel" are used interchangeable.

!n the context of the present invention, by "micro channels" or "capillary micro channel" is meant a very small narrow tube or channel through which a fluid can pass.

Preferably the diameter or with of a micro channel according to the invention is less than 1/5 of the capillary channel. Even more preferred the diameter of a micro channel according to the invention is less than 1mm, such as less than 0.5 mm, or less than 0.2 mm or even less than 0.1 mm- In a most preferred aspect the mircro channel has a diameter of 0,1 mm or less, e.g. 0.02-0.1 mm. The channels may also be formed of non-circular shapes, e.g. rectangular or triangular, in which case the "diameter" refers to the mean distance from the center of the cannel to the periphery.

Detailed description of the Invention

One object of the invention was to develop a capillary channel with enhanced drag force, and a device having such improved drag forces.

Another object of the invention was to develop a device for separation of fluid material into a retentate and a liquid fase without use of externa! driving force.

Accordingly in one aspect, the invention relates to a capillary channel having a width of 0.25-2,0 mm and height of 0.2-1 ,0 mm, characterised in that the capillary channel comprise one or more capillary microchannels,

Accordingly, In one aspect the invention relates to a device for separating a suspension comprising 200 μt or less into a liquid phase and a retentate phase, said device comprising an application chamber (1) comprising a hydrophilic filter material (17), said application chamber being connected to a capillary channel (3) according to the invention.

Another object of the invention was to develop a device for separation of red blood cells from plasma utilizing a single layer of filter material and a small volume of blood.

Therefore a device and a method was developed which were capable to separate whole-blood into a plasma/serum phase and a retentate phase (blood cells) in a short time, where the liquid phase is substantially free of retentate contamination, and where the separation is driven without the use of an external force.

It was observed that the presence of two anf three micro channels vere superior to the presence of one micrσchannel, and that specific dimensions were preferred. Accordingly, in a preferred embodiment the capillary channel has a width of 0,25-2.0 mm and height of 0.2-1.0 mm, and comprise two or more capillary microchannels. In one embodiment the lower part of the capillary channel has three or more capillary micro channels.

In one embodiment the width and height of the capillary micro channel(s) is 0.05-0.2 mm and 0.05-0,2 mm, respectively. In another embodiment the width and height of the capillary micro channel(s) is 0.08-0.12 mm and 0.08-0.12 mm, respectively.in another embodiment the width and height of the capillary micro channels(s) is about 0.10 mm and about 0.10 mm , respectively.

In another embodiment the invention relates to a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device comprising an application chamber (1) comprising a hydrophilic filter material (17), said ap- plication chamber being connected to a capillary channel (3) comprising microchannels as defined above.. These special capillary channels vere efficient in pulling the liquid into the capillary channel.

In a preferred embodiment the connecting junction between the application chamber and the capillary channel comprise a physical barrier (10, 10') preventing flow of residue retentate from a lower part of the chamber into the capillary channel. The presence of this physical barrier was surprisingly shown to create a substantially improved separation of the fluid material from the suspended matter. Accordingly, by visual inspection, it was observed that blood samples applied to the device without the physical bar- rier created a light red coloured fluid in the first capillary channel. However, when the connecting junction between the separation chamber and the first capillary channel comprised a physical barrier preventing flow of residue retentate from a lower part of the chamber into the first capillary channel, by visual inspection, it was observed that blood samples applied to the device created a transparent uncoloured fluid in the first capillary channel. In one embodiment the physical barrier is in the form of a vertical barrier having a height (10) of at least 0.2-1 -6 mm. in another embodiment the height (10) is at least 0.8-1.6 mm, In one embodiment the invention relates to a device, where the physical barrier 10) in the horizontal plane and in the direction towards the collection chamber describes an incline extending from the bottom of the application chamber. In one embodiment the incline in vertical direction is 0,2-1.6 mm, and in horizontal direction 0~ 100% of the length of the capillary channel In another embodiment the incline in vertical direction is about 0.8-1 ,6 mm, and in horizontal direction about 20-80% of the length of the capillary channel.

In one embodiment the device further comprises a collecting chamber (4) connected to the capillary channel.

In one embodiment the device comprises an upper part and a lower part, where the two parts when assembled form an application chamber (1), a capillary channel (3), where the lower part of the capillary channel has one or more capillary micro channels) (11 ) enhancing the capillary drag force of the capillary channel, and said upper part having an inlet leading to the application chamber. In one embodiment the interfaces between the upper and lower parts are sealed with a hydrophobic sealant.

In one embodiment the device further comprises a prefilter material (15).

In one embodiment the length of the capillary channel from the outlet of the application chamber to the inlet of collection chamber is 5 - 20 mm.

In another embodiment the invention relates to the use of one or more capillary micro channels as defined above, for enhancing the flow of liquid into a capillary channel.

In another embodiment the invention relates to the use of the device according to the invention for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, where the liquid phase is substantially free of suspended matter. The suspension might be blood.

in another embodiment the invention relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the sus- pended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying a suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; α. applying the filter material comprising the suspension to an application cham- ber, which is connected to a capillary channel as defined above; d. over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel

In another embodiment the invention relates to a method, where the liquid phase is di- rected into the capillary channel solely by the combined action of capillary forces provided by the capillary channel as defined above, and hydrostatic pressure generated by the applied sample.

In another embodiment the invention relates to a method where the blood is human blood.

In a preferred aspect the sample to be analysed preferably has a volume of less than 200μl. In an even more preferred aspect the sample to be analysed has a volume of less than 150μl, even more preferred less than 100μi, even more preferred less than 90μl, such as less than 80μl, less than 70μl or even less than 60μl. In an even more preferred aspect the sample to be analysed has a volume of less than 50μl, even more preferred less than 45μl, even more preferred less than 40μl.

In a preferred aspect the first part of the capillary channel has a volume of less than 10Oμl. In an even more preferred aspect the the capillary channe! has a volume of less than 90μl, even more preferred less than 80μl, even more preferred less than 70μl, such as less than 60μI, less than 50μl or even less than 40μl. In an even more preferred aspect the first part of the capillary channel has a volume of less than 30μt, even more preferred less than 25μl, even more preferred less than 20μl, such as less than 15μl, less than 10μl or even less than 5 μl. Example

Investigation of presence of physical barrier, corona treatment and micro channels on the separation into clear plasma in collection channel using blood filtra- tion device.

Conclusions

Presence of a physical barrier (10,) at the connecting junction between the separation chamber and the first capillary channel, preventing flow of residue retentate from a lower part of the chamber into the first capillary channel, result in an improved separation of the liquid and the suspended matter.

The corona treatment of at least the lower part of the internal surface of the first capil- lary channel facing the liquid, significantly enhances the filling of the collection chamber with plasma.

The use of micro channels in at least the lower part of the interna! surface of the first capillary channel facing the liquid is made of a surface treated plastic decreases the fill- ing time significantly.

Experimental setup

The blood filtration device used for the experiments was the milled K2 cartridge in clear polystyrene as illustrated in Fig. 2, with capillary stop and hydrophobic film covering the milled channels. The K2 blood inlet was used with oval 5 x 7.5mm pre-filter (vertical flow filter VF1, Whatman). The lateral flow filter 4x15 mm (Fusion 5, Whatman) was mounted on a hydrophobic adhesive. 100 μl K₃EDTA stabilized human blood (2 weeks old) was used for each experiment,

The volume of the collection chamber was 4.6 μl for the K2 device with the 3 micro channels («0.15x0.15mm).

The volume of the collection channel was measured by slowly filling it with indicator so- iution with a 1-10μl pipette.. The investigation was done using K2 cartridge as illustrated in Rg. 2 with and without the micro channels. For both setups the filling time of collection chamber for non corona treated and corona treated cartridges was measured.

Results

Preliminary investigations on the presence or absence of the physical barrier at the connecting junction between the separation chamber and the first capillary channel, preventing flow of residue retentate from a lower part of the chamber into the first capil- lary channel, showed an improved separation of the liquid and the suspended matter when the barrier was present

Further investigations on the capillary channels produced the following results: The volume of the collection chamber without the micro channels was measured to 3, 1 μl. The volume of collection chamber including the micro channels was 46 μl.

Corona treat- Micro chan- FiHing time (3.1μl) ment nels

No No Did not fill (5% after 12 min, plasma is accumulated at the tip of the filter but does not fully enter the collection chamber)

No Yes Did not fill (5% after 12 min, plasma is accumulated at the tip of the filter but does not fully enter the collection chamber)

Yes No 3.6 min

Yes Yes 2.6 min

Discussion

The results in the table above show it is very beneficial to corona treat the collection chamber in order to get it sufficiently hydrophilic and filled with plasma by capillary force. Note this is under the circumstances using hydrophobic film covering the milled channels. The table also shows a shorter filling time by the use of capillary micro channels milled in the capillary channel. The micro channels fills fast by capillary force and then promote the filling of the rest of the channel

Conclusion

The corona treatment is highly preferable to get the collection chamber filled with plasma.

The use of micro channels decreases the filling time signicantly

Claims

1. A capillary channel having a width of 0.25-2.0 mm and height of 0.2-1.0 mm, characterised in that the capillary channel comprise one or more capillary microchan- nels.

2, Capillary channel according to claim 1, where the width and height of the capillary micro channei(s) is 0.05-0.2 mm and 0.05-0.2 mm, respectively.

3. Capillary channel according to claim 2 where the width and height of the capillary micro channel(s) is 0,08-0.12 mm and 0,08-0.12 mm, respectively.

4. Capillary channel according to claim 3, where the width and height of the capillary micro channels(s) is about 0.10 mm and about 0,10 mm, respectively.

5. Capillary channel according to any of the preceding claims, where the lower part of the capillary channel has three or more capillary micro channels*

6. A device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device comprising a separation chamber (2) comprising an application zone (1) and a hydrophilic filter material (17), said separation chamber being connected to a capillary channel (3) according to any of claims 1-5,

7. Device according to claim 6, where the connecting junction between the applica- tion chamber and the capillary channel comprise a physical barrier (10) preventing flow of residue retentate from a lower part of the chamber into the capillary channel,

8. Device according to claim 7, where the physical barrier is in the form of a vertical barrier (10) having a height of at least 0.2-1,6 mm.

9. Device according to claim 8, where the height is at least 0.8-1.6 mm.

10. Device according to any of the claims 7-9, where the physical barrier (10) in the horizontal plane and in the direction towards the collection chamber describes an in- dine extending from the bottom of the application chamber.

11. Device according to claim 10, where the incline in vertical direction is 0,2-1.6 mm, and in horizontal direction 0-100% of the length of the capillary channel.

12. Device according to claim 11, where the incline in vertical direction is about 0.8- 1.6 mm, and in horizontal direction about 20-80% of the length of the capillary channel.

13. Device according to any of the claims 6-12 further comprising a collecting chamber (4a) connected to the capillary channel.

14. Device according to any of the claims 6-13 comprising an upper part and a lower part, where the two parts when assembled form a separation chamber (2) comprising an application well (V) and a hydrophilic filter (17), a capillary channel (3), where the lower part of the capillary channel has one or more capillary micro channel(s) (11) enhancing the capillary drag force of the capillary channel, and said upper part having an inlet leading to the separation chamber,

15. Device according to claim 14, where the interfaces between the upper and lower parts are sealed with a hydrophobic sealant.

16, Device according to any of the preceding claims, further comprising a prefilter material (15).

17. Device according to any of the preceding claims, where the length of the capillary channel from the outlet of the application chamber to the inlet of collection chamber is 5 - 20 mm..

18.. Use of one or more capillary micro channels as defined in any of claims 1-5, for enhancing the flow of liquid into the capillary channel,

19. Use of a device according to any of the claims 6-17, for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, where the liquid phase is substantially free of suspended matter.

20, Use according to claim 19, where the suspension is blood.

21. A method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying a suspension to a prefϋter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b, applying less than 200 μl of a sample suspension, or the liquid of step a., to a fiiter material; c. applying the filter material comprising the suspension to an application chamber, which is connected to a capillary channel as defined in any of claims 1-5; d. over saturating the fiiter to feed the capillary channel; e. directing the liquid phase into the capillary channel.

22. Method according to claim 21, where the liquid phase is directed into the capillary channel solely by the combined action of capillary forces provided by the capillary channel as defined in any of claims 1-5 and hydrostatic pressure generated by the applied sample.

23, Method according to claim 18, where the blood is human blood.