WO2013160668A2

WO2013160668A2 - Miniaturised centrifugation apparatus

Info

Publication number: WO2013160668A2
Application number: PCT/GB2013/051027
Authority: WO
Inventors: Andrew Paul DIXON; Andrew John BOWYER; Miles Warwick ASHCROFT
Original assignee: Magna Parva Limited
Priority date: 2012-04-24
Filing date: 2013-04-23
Publication date: 2013-10-31
Also published as: GB2501615A; GB201307344D0; WO2013160668A3; GB201207178D0

Abstract

A centrifuge comprises a casing (2) and a rotor (1) mounted on a bearing (4) for rotation inside the casing. Inside a circumferential wall (12) of a bowl of the rotor (1) there are arranged in an axial sequence: a settlement zone where fluid can collect, an annular weir (13) projecting radially inwards, and an escape zone (14) where fluid can flow out of the bowl. The escape zone may comprise a set of holes (15) through the wall (12) in the base of an annular gulley (14). The bowl of the rotor (1) rotates in a chamber of the casing (2) and an annular seal (5) between the rotor (1) and the casing (2) divides the chamber into two parts (17, 18) to prevent fluid from reaching the bearing (4). Air ducts (19) may connect the two parts of the chamber to equalize the pressure between them. The centrifuge can be manufactured as a small, disposable unit designed to work with an external drive.

Description

Title

Miniaturised Centrifugation Apparatus Field of the invention

The present invention relates to centrifuges and centrifugation devices or separators, particularly those employed in the preparation of biological or chemical samples in automated processing or analysis systems, but may find application in any application where substances of different densities or phases must be separated, clarified, mixed, agitated, homogenised, disrupted or otherwise exposed to a given acceleration environment.

Prior Art

In the current state of the art, laboratory-type centrifuges generally employ spinning rotors into which a number of small vials or tubes may be placed. Such systems require either manual action or a robotic device to load and unload and are therefore suited to the processing of large numbers of small samples. Industrial and pharmaceutical spinning-bowl centrifuges use a large bowl, into which the process fluid is fed. The suspended matter forms a compacted mass against the walls of the bowl, whilst the supernatant is removed at the top. This type of apparatus is suited to large batches or continuous operation. Large, industrial-scale centrifuges cannot simply be reduced proportionately to laboratory scales because of the different flow regimes and the effects of velocity and Reynolds number that occur at such smaller scales. Smaller spinning-bowl centrifuges are known, which employ disposable or cleanable bowls and are respectively used for blood plasma separation and lubricating oil cleaning. Representative patents include:

UK Pat. Appln. No. GB 2475835 (A) discloses a sample processing system which employs modules to perform various functions such as reagent addition, thermo-cycling, filtration and centrifuging. The device has a base unit which contains driving means for all such modules such that no expensive components such as motors, actuators etc. need to be contained within the modules, rendering them cheap and disposable. Thus the prior art shows a need for a disposable miniaturised centrifuge for use in modular sample processing apparatus which can be used to process sample volumes ranging from about 0.5ml to about 50ml.

I nter national Pat. Appln. No. WO 2009/ 131659 (A1 ) discloses a centrifuging means whereby a disposable rotating bowl is employed to rotate a body of fluid such that suspended solids may be separated out. The centrifuging means is designed for processing batches of fluid from 300 to 2000 litres in volume and is provided with an aseptic means for feeding fluid into and supernatant out of the bowl. Thus the prior art shows the use of a rotating bowl as a centrifuge, which can process a greater volume of fluid than the internal volume of the bowl.

US Pat. No. 4086924 (A) discloses a means for extracting blood plasma from a donor by means of an automatically-controlled spinning-bowl type centrifuge. US Pat. No. 3567113 (A) discloses a portable centrifuge device for performing haemocrit and other such analyses on samples of blood in situations where no electrical power or laboratory facilities are available. Thus the prior art shows a need to centrifuge samples in a non- laboratory setting.

US Pat. No. 5656164 (A) discloses a centrifugation device for cleansing oil in engines, particularly marine engines, by means of a spinning bowl into which the

oil/water/ contaminant mixture is deposited. Centripetal force causes the contaminants and water to be separated from the oil. This device, although presented with the intention of providing a compact means of centrifugally separating fluids of different densities and suspended particles, is still too large for convenient operation in portable equipment with sample sizes below about 30ml.

US Pat. No. 63984202 (B1) discloses a handheld sample homogeniser device for the preparation of biological samples in a laboratory. The homogeniser employs a close-fitting set of cylindrical rotor and stator blades which are immersed in a principally liquid sample. Shear forces and rotor-sample interactions including cavitation cause cells within the sample to be disrupted. Thus the prior art shows that rotor-stator homogenisers are suitable for the disruption of cells in biological samples.

Statement of the Invention

The invention provides a centrifuge as defined in claim 1. Preferred but non-essential aspects of the invention are defined in the dependent claims. The present invention provides a means whereby the aforesaid problems may be addressed by a disposable centrifuge, reducing the possibility of cross-contamination between samples. The invention allows for both batch and continuous-flow modes of operation, enabling a relatively small device to process a wide range of sample/reagent volumes. Furthermore the invention is intended to be installed in a sample processing device as a replaceable single- use component so does not require time-consuming cleaning operations, reducing the cost of sample preparation. The sample processing device also provides a drive for the rotor and optionally also provides means for the automated supply and removal of fluid so the centrifuge unit does not require expensive components such as motors and pumps. The invention enables the cost-effective packaging of a sample preparation means along with an analysis means such as a lateral flow chip or antibody assay so that samples may be prepared and analysed in one module. In an exemplary embodiment of the invention, these and other objects are met by having a nominally cylindrical bowl, to which is fitted a hollow shaft. The shaft runs within a bearing arrangement, which is suitably sealed to prevent the ingress of liquids. Disposed about the cylindrical bowl is a casing which incorporates a close-fitting lip. The lip may be formed in sliding contact with the outer surface of the bowl or in contact with the outer surface of the hollow shaft between the bowl and the bearing. The lip may be manufactured from an elastomeric material or may be formed from the same material as the casing for example. Holes are located about the periphery of the bowl in order to provide an exit path for the centrifuge supernatant. The interior profile of the bowl incorporates a beach or weir comprising a substantially conical or ogive-shaped ramp where the rotor diameter is reduced such that the process fluids are trapped in a settlement zone of the rotor bowl, allowing suspended matter to settle out and compact against the rotor wall. As more fluid is added the supernatant will spill over the weir and is ejected from the spinning rotor via the radial holes in the rotor. The supernatant is collected by the centrifuge casing and drains into an outflow port. Rotational motion is supplied to the rotor via a gear and drive means such as an electric motor which is contained within the 'parent' processing system, which engages with a toothed portion of the rotor. Other mechanical drive means such as a drive belt or a friction drive are possible. In this way the entire disposable centrifuge unit comprises a minimum quantity of components and, within a radius of about 25mm, may generate acceleration forces in the fluid of at least 2500g by rotating at speeds of about 10000 RPM.

In a further exemplary embodiment of the invention, a stator is incorporated into the assembly within the rotor bowl. This refinement allows for more efficient agitation and mixing to be carried out. Furthermore, if the rotor is manufactured to incorporate blade-like features, which move in close proximity to similar features on the stator, the maceration or homogenisation of tough sample material may be accomplished. This method is also suitable for the disruption of cellular samples, spores and the like. Disruption-promoting media such as glass beads in the size range 10-500μΓη may be used with such a scheme, as is the current practice in laboratory-based apparatus. Furthermore reagents such as lysis agents, solvents and surfactants may be pre-loaded into the rotor bowl. These may be in the form of liquids or may alternately be dehydrated for easier transport and storage and require the addition of water or other solvents when loading the sample into the rotor. An ultrasonic horn could be built into the centrifuge unit or into the sample processing device to enhance the disruption of cells in the sample.

In another exemplary embodiment of the invention the cylindrical rotor is provided with a number of permanent magnets, or alternatively a number of electrical windings which are arranged about its periphery. A number of magnetic coils disposed either within the casing or external to the centrifuge unit are then energised such that rotational motion is induced in the rotor. This embodiment has the advantage that no demountable drive is required, reducing the number of component parts and reducing the number of sealing elements required.

In another exemplary embodiment of the invention, pumping features are incorporated into the rotor; these may take the form of substantially disc-shaped members, which throw the ejected supernatant into an annular collection channel or may use helical grooves in the rotor outer surfaces, such that any droplets on the rotor or casing walls are driven toward the outflow duct. A yet further alternative pumping means may employ a series of vanes similar to the arrangements used in state of the art centrifugal pumps, to force the ejected supernatant into an outflow duct. In an alternative exemplary embodiment of the invention, the assembly is entirely or partially reusable and manufactured such that it may be cleaned or sterilised a number of times. This embodiment has the advantage that very little waste material is generated with each sample preparation and that the individual components are easily replaceable.

In an alternative embodiment of the invention, the disruption stator blades are located partway up the rotor wall and additional weir and catchment features are positioned within the rotor. In this way the invention may be used to first separate or clarify a sample, then perform a cell disruption followed by further clarification by rotating the rotor at the appropriate speeds. For example, if the stator is located such that its blades lie halfway up the rotor, rotation at a moderate speed will cause dense material to be separated from the sample. If the rotor is then rotated at a higher speed, the clarified sample will come into contact with the stator blades, and is thereby homogenised. Rotating the rotor at a still greater rate will caused the clarified, homogenised sample to be ejected from the rotor in the manner described above. This embodiment is advantageous in that it allows two processes to be accomplished in one module, and could be used, for example when it is required to analyse the contents of white blood cells. In this case the red cells must be removed before the white cells are homogenised.

An advantage of all embodiments taught herein is that the centrifugation apparatus may be made very compact and from inexpensive materials, as all drive means are provided by an external apparatus. Therefore a centrifuge manufactured according to the invention may be disposable and compatible with a sample processing system of the type disclosed in

GB2475835 (A) - Sample Processing System.

It is a further advantage of the present invention that the layer of fluid trapped in the centrifuge rotor is thin, and the rotor may be made to rotate very quickly because it has a low mass. Therefore the settling velocity of suspended particles within a given fluid will be short compared to that in samples which undergo centrifugation by conventional means. As can be seen in equation 1, a form of Stokes' Law, the effect of miniaturisation is not detrimental to the aim of producing a high acceleration environment because the effect of rotor angular velocity on the settling velocity is squared compared to the linear relation between rotor radius and settling time.

Equation 1

Where ins the settling velocity, Δρ is the difference in density between solid and liquid components, (lis the solid particle diameter, ns the rotor radius, wis the rotor angular velocity and μϊ≤ the liquid viscosity.

Brief description of the Drawings

Fig. 1 depicts a simplified cutaway schematic of an exemplary embodiment of the invention. Fig.2 depicts a simplified cutaway schematic of the rotor, seal and bearing according to the exemplary embodiment of the invention shown in Fig. 1.

Fig.3 depicts a simplified cutaway schematic of the exemplary embodiment of the invention shown in Fig. 1, with regions of low and high air pressure identified.

Fig.4 shows a sectional view of a second embodiment of the invention.

Fig.5 shows a perspective view of a centrifuge rotor as employed in a third embodiment of the invention.

Fig.6 shows in a sectional view on a plane perpendicular to the axis, a schematic representation of a fourth embodiment of the invention.

Specific Description

Fig. 1 shows a sectional view of a preferred embodiment of the present invention. The rotor (1) is carried within the case (2) and comprises a bowl and a lid that together define a rotor chamber (3). A hollow shaft protrudes from the lid and is fixed to one race of a sealed-type ball bearing (4) , the other race of which is fixed to the case (2) . A lip-seal (5) prevents leakage of the supernatant to the region of the bearing (4). Rotational movement is imparted to the rotor (1) by means of a gear wheel (6) that forms part of an external system and engages with gear teeth (not shown) on an outer surface of the rotor ( 1 ) . A stator (7) is suspended within the rotor ( 1 ) by means of a support shaft (8) that passes through the hollow shaft of the rotor ( 1 ) and is fixed to a plate (9) on the exterior of the case (2). The stator (7) promotes homogenisation or cell disruption through fluid shear forces and direct cell-stator interaction. Vanes (10) formed as part of the rotor ( 1 ) serve to further promote homogenisation or disruption.

Fig.2 shows a sectional view of the rotor ( 1 ) and stator (7) of the preferred embodiment of the present invention. The process fluid or suspension is introduced into the centrifuge (which is preferentially already rotating at a slow speed of between about 200RPM and

2000 RPM) via an orifice (11) through the centre of the support shaft (8) of the stator (7) and is allowed to fall under the action of gravity into the rotor chamber (3). When the fluid comes into contact with the slowly rotating rotor bowl (1), it will be driven against and through vanes of the stator (7) causing cell disruption and mixing it via shearing forces in the fluid. The direction of rotation of the rotor may be periodically reversed to agitate the fluid in the bowl. In order to separate denser solid matter, for example disrupted cell matter or soil, from the sample, the rotor (1) is rotated at a higher speed (greater than about 7000RPM) causing centripetal forces to act on the fluid whereby it forms a layer on the inside wall (12) of the rotor (1), where suspended matter will settle out. As more fluid is added, the thickness of the fluid layer will exceed the height of the weir (13) and clarified supernatant will spill over into a collection gulley ( 14) around the inside surface of the rotor bowl. Holes (15) in the gulley (14) floor allow the supernatant to exit the rotor (1).

Fig.3 shows a further sectional view of the preferred embodiment of the present invention. In this embodiment, a low friction seal between the case (2) and the rotor (1) is achieved by employing a lip seal (5) which has a diameter larger than that of the rotor (1) by about 50 to 100 microns. When the rotor ( 1 ) is spinning, a low pressure zone (16) is generated in the gap between the lip seal (5) and the rotor ( 1 ) due to the layers of fast -moving air in this location. In order to prevent the supernatant from being drawn into the low pressure zone (16) and leaking out around the seal, relatively high pressure air pressure in the zone above (17) the lip seal (5) is allowed to flow through the seal gap into the low pressure zone below ( 18) the seal (5) . The air is allowed to circulate via air ducts ( 19) and openings in the casing (2), which allows gravity to overcome the force generated by the low pressure zone (16) and the supernatant to fall into an outflow duct (20) in the base of the case (2) . The inside walls of the centrifuge chamber may be provided with guide channels (not shown) to guide liquid flow towards the outflow duct (20). The inside walls may also be provided with a hydrophobic surface to reduce adhesion between the walls and the supernatant fluid (which is typically aqueous). Fig.4 shows a sectional view of an alternate embodiment of the invention. In this embodiment the seal between the rotor (21) and the case (22) is achieved by placing a lip seal (23) within the case (22) such that it bears on the rotor drive shaft (24). In this way the friction resulting from the seal is minimised thus reducing the energy required to operate the centrifuge as well as the wear rate of the seal (23). The shaft (24) is provided with a gear wheel (25) which is used to couple rotational motion to the assembly from an external electric motor (not shown). Ball bearings (26) are used to support the shaft (24) and are preloaded by means of a wave-spring (27). To promote the transfer of fluid into the rotor (21 ) , a tube (28) is provided which feeds liquid directly into the centrifuge rotor (21 ) . Operation of the centrifuge is identical to that discussed in the previous embodiment;

however the supernatant is extracted from the rotor (21 ) via a series of ducts (29) in the outer wall of the rotor (21). The ducts are angled outwards from the floor of the collection gulley and in this embodiment they emerge through the lower end wall of the rotor. This reduces the loss of supernatant that may adhere in the form of droplets to the walls of the centrifuge chamber (30) by directing the output supernatant toward the outlet port (31) of the centrifuge.

Fig.5 shows a view of a centrifuge rotor (32) as employed in a further, alternate embodiment of the invention. The rotor (32) is provided with a series of vanes (33) that allow the rotor (32) to be used as a pump, which can be advantageous in transferring larger quantities of supernatant out of the centrifuge assembly. Additionally the vanes (33) serve to detach droplets of supernatant that have emerged from the ejection ports (34) of the rotor (32) but which remain attached to the outer wall of said rotor (32).

Fig.6 shows a schematic representation of a further embodiment of the invention. In this embodiment, the rotor (35) has a number of permanent magnets (36) embedded within it and arranged to present a magnetic field of alternating polarity about the periphery of the rotor (35) . A number of electromagnetic coils (37) are disposed about the centrifuge chamber such that by providing a varying current to the terminals (38) of the coils (37), the rotor (35) can be caused to rotate. The speed and direction of rotation can be altered by changing the oscillation rate of, and the phase between, the electrical signals applied to the terminals (38) of the individual coils (37). This embodiment is advantageous as it dispenses with complicated and expensive driving mechanisms such as gears, belts and chains, allowing the centrifuge chamber to be fully sealed. The above description of the preferred embodiment has been given by way of an example. From the disclosures given, those skilled in the art will understand the invention and its advantages, and will also find apparent changes and modifications to the structures and processes disclosed. It is sought therefore to cover all such changes that lie within the scope of the invention, as defined in the appended claims and equivalents thereof.

Claims

1. A centrifuge comprising:

a casing (2, 22) ;

a rotor (1, 21, 32, 35) mounted on a bearing (4, 26) for rotation inside the casing

(2, 22), the rotor comprising:

a bowl defined by an end wall and a circumferential wall;

a settlement zone where fluid in the bowl can collect on an inner surface of the circumferential wall when the rotor (1, 21, 32, 35) is rotating;

an escape zone (14) where fluid can flow from the interior to the exterior of the bowl when the rotor ( 1 , 21 , 32, 35) is rotating; and

an annular weir (13) projecting radially inwards from the circumferential wall to divide the settlement zone from the escape zone (14).

2. A centrifuge according to claim 1 , wherein a surface of the weir (13) adjacent to the settlement zone is in the form of a ramp.

3. A centrifuge according to claim 1 or claim 2, wherein the settlement zone is located between the weir ( 13) and the end wall of the bowl, and wherein the escape zone (14) is located on the side of the weir (13) that is remote from the end wall.

4. A centrifuge according to any preceding claim, further comprising one or more fluid passages (15, 29) in the circumferential wall of the rotor bowl, each fluid passage having an entrance hole in the escape zone (14).

5. A centrifuge according to claim 4, wherein the escape zone ( 14) further comprises an annular gulley, in the base of which the entrance holes are located.

6. A centrifuge according to any preceding claim, wherein the casing (2) comprises a chamber in which the bowl of the rotor (1) rotates, the centrifuge further comprising an annular seal (5) between the casing (2) and the rotor ( 1 ) , the seal (5) dividing the chamber into a first part ( 17) adjacent to the bearing (4) and a second part

( 18) into which fluid flowing from the escape zone (14) of the bowl is discharged.

7. A centrifuge according to claim 6, further comprising one or more air ducts (19) connecting the first and second parts (17, 18) of the chamber.

8. A centrifuge according to claim 6 or claim 7, further comprising vanes (33) on an exterior wall of the rotor (32) for transferring fluid onto an inner wall of the chamber.

9. A centrifuge according to any of claims 6 to 8, further comprising a hydrophobic surface on an inner wall of the chamber for promoting the flow of aqueous fluid towards a discharge port (20, 31).

10. A centrifuge according to any preceding claim, wherein the rotor (1, 21, 32, 35) further comprises a lid that is sealed to the bowl, whereby the interior of the bowl is enclosed except at the escape zone ( 14) and at an inlet aperture (11, 28) .

11. A centrifuge according to claim 10, wherein the lid of the rotor comprises an axle (8, 24) by which the rotor (1 , 21 , 32, 35) is mounted on the bearing (4, 26), the inlet aperture (11, 28) being provided by a bore through the axle.

12. A centrifuge according to any preceding claim, wherein the rotor (1 , 21 , 32, 35) is provided with blades, ridges, vanes or paddles for agitating fluid in the bowl.

13. A centrifuge according to any preceding claim, further comprising a stator (7) that is fixed to the casing (2) and is suspended inside the bowl of the rotor (1).

14. A centrifuge according to claim 13, wherein the stator (7) is provided with blades, ridges, vanes or paddles for agitating fluid in the bowl.

15. A centrifuge according to claim 13 or claim 14, wherein the stator (7) is suspended on a hollow shaft (11) that provides a conduit for introducing fluid into the bowl of the rotor ( 1 ) .

16. A centrifuge according to any of claims 1 to 15, wherein the rotor ( 1 ) further

comprises drive means for mechanically coupling the rotor (1) to an external drive (6).

17. A centrifuge according to any of claims 1 to 15, wherein the rotor (35) further

comprises a set of permanent magnets (36) or electrical coils, whereby rotation of the rotor (35) can be induced by a rotating electromagnetic field from an external drive (37) .

18. A centrifuge according to any preceding claim, wherein the rotor (1 , 21 , 32, 35) is irreversibly secured in the casing (2).

19. A centrifuge according to any of claims 1 to 18, wherein the rotor (1 , 21 , 32, 35) rotates about a substantially vertical axis. A centrifuge according to any of claims 1 to 18, wherein the rotor (1, 21, 32, 35) rotates about a substantially horizontal axis.