WO2013045631A1

WO2013045631A1 - Method and device for producing fluidically separated sub-volumes of a liquid

Info

Publication number: WO2013045631A1
Application number: PCT/EP2012/069217
Authority: WO
Inventors: Maximilian Focke; Felix Von Stetten; Oliver Strohmeier
Original assignee: Albert-Ludwigs-Universität Freiburg; Hahn-Schickard-Gesellschaft für angewandte Forschung e.V.
Priority date: 2011-09-30
Filing date: 2012-09-28
Publication date: 2013-04-04
Also published as: DE102011083920B4; DE102011083920A1

Abstract

Fluidically separated sub-volumes of a liquid are produced within a carrier comprising a fluidics structure which has a distribution channel and fingers which branch off from the distribution channel and define a specific volume, respectively. Each finger is fluidically connected to a chamber. By rotating the carrier, the fingers are filled with the liquid via the distribution channel, so that the fingers have liquid sub-volumes contained therein, gas volumes or aerosol volumes occluded within the chambers preventing the liquid from getting into the chambers from the fingers. The temperature of the gas or aerosol within the chambers is reduced, so that an under pressure results within the chambers which causes the liquid sub-volumes to be drawn into the chambers from the fingers.

Description

Method and Device for Producing Fluidically Separated Sub-volumes of a Liquid

Description The present invention relates to methods and devices for producing fluidically separated sub-volumes of a liquid and, in particular, to such methods and devices which are suitable for centrifugal-microfluidic platforms.

Centrifugal-microfluidic systems deliberately employ centrifugal forces for transporting liquids through channels on a rotating carrier. In this context, the channels have typical dimensions within the micrometer to millimeter ranges and enable transporting liquids having volumes within the nanometer to milliliter ranges.

In order to be able to realize relatively complex fluidic processes it is necessary in most cases to integrate valves into the microfluidic structures. To reduce cost and complexity, passive valves are preferably used, changes in the centrifugal force being specifically exploited for switching liquids. An overview of passive valve concepts on centrifugal- microfluidic platforms is given in D. Mark et al, "Microfluidic Lab-on-a-Chip platforms: requirements, characteristics and applications", Chem.Soc.Rev., 2010, 39, p. 1 153-1 182. In order to change the centrifugal force acting upon a liquid given a constant mass and a radial position on the rotating carrier (test carrier) it is necessary, as a matter of principle, to be able to change the rotational speed of the test carrier. However, depending on the equipment used for processing the rotating carrier and on the application, it is not always possible to influence the rotational speed of the carrier.

Typical fields of application of centrifugal microfluidics include the life sciences and medical diagnostics. Centrifugal microfluidics here offer the possibility, among other things, of automating, miniaturizing and parallelizing standard laboratory procedures by means of application-specific microfluidic carriers, which may be referred to as test carriers. An essential step in a multitude of such standard laboratory procedures is to split up an initial amount of liquid into several sub-volumes, which is referred to as aliquoting; the sample typically being a substance in the liquid phase. Following said splitting up, each sub-volume (aliquot) may be subjected to a specific test on the test carrier. Said automatic aliquoting of the initial amount of liquid into several sub-volumes thus enables determining several diagnostically relevant parameters from a shared sample in parallel and is the prerequisite of said parallel determination. Such parallel determination is typically referred to as "panel testing", which means parallel determination of several diagnostically relevant parameters, which typically are grouped in a sensible manner. Prior Art

Centrifugal-microfluidic systems are known that exploit variable centrifugal forces in order to aliquot an initial amount of liquid. Typically said systems enable aliquoting and subsequent transferal to fluidically separated structures via a corresponding valve structure or other fluidic retention structures. However, variable rotational speeds are always required for this purpose.

For example, centrifugal-microfluidic systems for aliquoting at variable and adjustable rotational speeds while using back-pressure valves in order to aliquot an amount of liquid into a plurality of fluidically separated chambers are known from D. Mark et al., "Aliquoting on the centrifugal microfluidic platform based on centrifugo-pneumatic valves", Microfluidics and Nanofluidics, 10, p. 1279-1288, 201 1 ; D. Mark et al„ "Centrifugo-pneumatic valve for metering of highly wetting liquids on centrifugal microfluidic platforms", Lab Chip, 9, p. 3599-3609, 2009; and DE 102008003979 B3. At first, an initial amount of liquid is directed through a microfluidic distribution channel at a frequency fl . A plurality of fluidic finger structures having defined sub- volumes are fluidically connected to said distribution channel and face the radially outer side of the rotating test carrier. While flowing through the distribution channel, the liquid will fill said fluidic finger structures on account of the centrifugal force, whereas the supernatant liquid is transferred from the distribution channel to an excess chamber. Each fluidic finger is connected, on the radially outer side, to an unvented fluidic chamber via a fluidic channel. At the rotational speed f 1 , the occluded air exerts a sufficiently high back pressure on the liquid within the fluidic finger so as to prevent it from flowing into the unvented chamber on account of the centrifugal force. A cyclic change in the rotational speed between a rotational speed f2 > f 1 and a rotational speed fl , however, results in that the liquid from the fluidic finger structures is pushed in, in portions, among the air and into the unvented chambers. Aliquoting while using other fluidic retention structures is described in US 7300199 B2, in N. Honda et al, "Simultaneous multiple immunoassays in a compact disc-shaped microfluidic device based on centrifugal force", Clin Chem 51(10), p. 1955-1961, 2005, as well as in WO 2004/083108 Al . Said documents describe utilization of geometric valves, possibly with a hydrophobic surface coating, a microfluidic aliquoting structure on a rotating test carrier consisting of a zigzag-shaped channel arranged along a radius. In particular, said documents describe microfluidic structures wherein a distribution channel is initially filled in a capillary manner with the liquid to be aliquoted. Here, a sufficiently low rotational speed fl is necessary in order to enable capillary filling counter to the centrifugal force. The distribution channel is arranged in a zigzag-shaped manner such that the channel exhibits radially inner areas which are vented and radially outer areas which are in fiuidic connection with downstream structures via a liquid retention structure. In this context, the volume of the aliquot is defined by the channel volume between two radially inner, adjacent air vents in each case. In order to transfer the sub- volumes to separate chambers, a rotational speed f2 > f 1 is subsequently required in order to surmount the liquid retention structures.

Moreover, systems are known for splitting up an amount of liquid at a constant rotational speed wherein the individual sub-volumes are not fluidically separated, however, so that there is an increased risk of cross contamination. Such systems are described in C. Schembri et al., "Centrifugation and capillarity integrated into a multiple analyte whole blood analyser", Journal of Automatic Chemistry, vol. 17, p. 99-104, 1995, and in US 6752961 B2. Here, too, fiuidic finger structures are connected to a distribution channel on its radial exterior surface. However, said finger structures are not fluidically connected to further separate chambers. The individual fingers are filled, via the distribution channel, with a defined sub-volume of an initial amount of liquid; however, no complete fiuidic separation of the individual sub-volumes takes place. K. Abi-Samra et al, "Thermo-pneumatic Pumping in Centrifugal Microfluidic Platforms", Microfluidics Nanofluidics (published online on June 17, 201 1), 201 1, describes application of the thermo-pneumatic effect in connection with centrifugal-microfluidic systems in order to transfer a liquid from a radially outer chamber to a radially further inward chamber counter to the centrifugal force. In this context, liquid is located within a microfluidic reservoir on a centrifugal-microfluidic platform. An unvented chamber is connected to the microfluidic reservoir. By increasing the temperature in the unvented chamber, the air contained therein expands and pushes the liquid out of the microfluidic reservoir into a radially further inward chamber. From US 2008/0149190 Al a centrifugal-microfluidic platform is known wherein by means of locally heating and subsequently cooling a thermal transfer structure an analyte may be transported, in portions, from a microfluidic chamber into a second microfluidic chamber formed by the thermal transfer structure. This may possibly involve cyclic repetitions of heating and cooling. In accordance with US 2008/0149190 Al a filling chamber is connected to a first chamber via a channel extending radially outward. The first chamber has an analyte located therein, said first chamber being connected to thermal transfer structures via channels. A fluid within the thermal transfer structures is heated via a heating structure and expands into the first chamber. Following subsequent cooling of the fluid, some of the analyte is drawn into the thermal transfer structures.

The object underlying the present invention consists in providing methods and devices which enable producing sub-volumes of a liquid, which are completely separated from one another fluidically, without requiring different rotational speeds; no risk of cross contamination must exist during and following said production of the sub-volumes.

This object is achieved by a method as claimed in claim 1 and by a device as claimed in claim 12.

Embodiments of the invention provide a method of producing fluidically separated sub- volumes of a liquid within a carrier comprising a fluidics structure having a distribution channel and fingers branching off from the distribution channel and defining a specific volume, respectively, each finger being connected to an unvented chamber, and each finger having a course, in relation to a center of rotation, which radially falls from the distribution channel to the chamber to which the finger is connected, the method comprising: filling the fingers with the liquid via the distribution channel by rotating the carrier, so that the fingers have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers preventing the liquid from getting into the chambers from the fingers; and reducing the temperature of the gas or of the aerosol within the chambers, so that an underpressure will result within the chambers which causes the liquid volumes to be drawn into the chambers from the fingers.

Embodiments of the invention provide a device for producing fluidically separated sub- volumes of a liquid, comprising: a carrier comprising a fluidics structure having a distribution channel and fingers branching off from the distribution channel and defining a specific volume, respectively, each finger being connected to an unvented chamber, and each finger having a course, in relation to a center of rotation, which radially falls from the distribution channel to the chamber to which the fmger is connected; a drive means configured to cause the carrier to rotate so as to rotate the carrier around the center of rotation so as to fill the fingers with the liquid via the distribution channel, so that the fingers have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers preventing the liquid from getting into the chambers from the fingers while the fingers are being filled with the liquid; a heating means configured to temper a fluid within the chambers; and a control means configured to control the heating means to heat a gas or aerosol within the chambers to a first temperature prior to the fingers being filled with the liquid, and control the heating means to reduce, following filling of the fingers, the temperature of the gas or aerosol within the chambers to a second temperature, which is lower than the first temperature, so that an underpressure will result within the chambers which causes the liquid volumes to be drawn into the chambers from the fingers. filling the fingers with the liquid via the distribution channel by rotating the carrier, so that the fingers have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers preventing the liquid from getting into the chambers from the fingers; and reducing the temperature of the gas or of the aerosol within the chambers, so that an underpressure will result within the chambers which causes the liquid volumes to be drawn into the chambers from the fingers.

Embodiments of the invention are based on the finding that given a constant rotational speed, aliquots may be produced from an amount of liquid and may be stored in a fluidically completely separated manner in that centrifugo-thermopneumatic switching in connection with a microfluidic structure is used for aliquoting an initial amount of liquid into separate fluidic chambers. In this context, hermopneumatic switching is understood to mean switching wherein pressure differences induced by temperature differences are exploited, in a microfluidic system, to move and switch liquids. In embodiments of the invention, the thermopneumatic force in the radial direction may be directionally amplified by means of additional rotation of the microfluidic system, which may be referred to as centrifugo-thermopneumatics.

In embodiments of the invention, the rotational speed of the carrier is kept constant while the fingers are being filled and while the temperature of the gas or of the aerosol within the chambers is reduced, so that no drive means is required whose rotational speed is variable or adjustable.

In embodiments of the invention, prior to aliquoting, i.e. to filling the fingers, the temperature is increased so as to put a gas within the chambers into an expanded state so as to contract accordingly upon subsequent reduction of the temperature to cause the liquid to be drawn into the chambers. In embodiments of the invention, the gas within the chambers may be air. In embodiments of the invention, prior to aliquoting, i.e. to filling the fingers, the temperature is increased to increase the temperature of an aerosol within the chambers; upon subsequent reduction of the temperature, the aerosol will condense and contract accordingly so as to cause the liquid to be drawn into the chambers. The back pressure which builds up while the fingers are being filled and which prevents the liquid from getting into the chambers from the fingers may be largely independent of the temperature, given a constant temperature,.

In embodiments of the invention, the fingers are filled via a distribution channel having a radially falling azimuthal course, so that at a radially inner end of each finger, shearing off of the liquid takes place and the distribution channel is emptied into an overflow chamber fluidically connected to a discharge end of the distribution channel. Thus, it is possible for each finger to separately have a defined liquid sub-volume present therein. The chambers are unvented chambers. In this context, an unvented chamber is understood to mean a chamber which allows no escaping of the gas or aerosol from the chamber. It may be an end chamber which is closed except for the connection to the fingers. In alternative embodiments, at least some of the chambers may be connected to a further, unvented chamber. In alternative embodiments, the chambers may have closable openings that are closed while the fingers are being filled, so that they are unvented chambers and so that the pneumatic effect described occurs.

In embodiments, a fluidic connection between the fingers and the chambers may be formed by a connecting channel whose flow cross section is smaller than a cross section of the fingers and of the chambers transverse to the flow direction.

The length of the connecting channels along the radially falling distribution channel may decrease, so that the chambers along a radius are arranged at an identical radial distance from the center of rotation. This enables tempering of the chambers and/or of a fluid within the chambers while using a local heating means arranged at an identical radial distance from the center of rotation as are the chambers. Thus, the chambers and/or the fluid within the chambers may be locally heated in a simple manner.

In embodiments of the invention, the chambers and the gas or aerosol located therein are heated locally. In embodiments of the invention, the entire carrier is heated globally by means of a corresponding heating means; due to the chambers being unvented, pressure differences will arise as a result of which the liquid will be drawn into the chambers.

Embodiments enable controlling and, in particular, aliquoting liquids with the aid of a centrifugo-thermopneumatic switching principle. Embodiments of the invention thus relate to centrifugal-microfluidic systems - a centrifugal-microfluidic system may be understood to mean a rotating carrier (test carrier) which has microfluidic structures introduced therein and which may be disc-shaped, for example, or may have the shape of part of a disc, e.g. the shape of a slice of cake or of a wedge. Embodiments of the invention relate to a microfluidic structure on a rotating carrier in connection with a switching concept for handling a liquid in the rotating carrier, which is suited, in particular, to aliquot an initial amount of liquid into a plurality of sub-volumes derived therefrom, and, in particular, to subsequently transfer same from a first chamber (fingers) to a second chamber in a defined manner without having to change the rotational speed of the centrifugal-microfluidic system for this purpose. The first chamber (fingers) may be radially closer to the center of rotation than the second chamber, so that centrifugal amplification of the thermopneumatic force in the radial direction may take place while the defined liquid sub-volumes are transferred from the first chamber to the second chamber. In alternative embodiments, the second chamber may be radially closer to the center of rotation than the first chamber (fingers), in which case the thermopneumatic force may be sufficient to overcome the centrifugal force caused by the rotation. Transferring the aliquots into a second chamber may cause fluidic separation thereof, so that cross contamination between the tests performed within the second chambers with the aliquots may be avoided.

Thus, embodiments of the invention enable aliquoting of an initial amount of liquid without changing the rotational speed of the rotating carrier as is necessary in the operation of speed-controlled valves. By transferring the aliquots from the fingers to the chambers, the risk of cross contamination via liquid bridges, which otherwise may form between the individual fluidic fingers, may be avoided. Embodiments of the invention thus allow integration of centrifugal-microfluidic systems into established laboratory equipment such as centrifugal thermocyclers, for example. In this connection, in embodiments of the invention, after splitting the aliquots are transferred to separate chambers and are present in a state in which they are completely separated fluidically.

Therefore, embodiments of the invention provide a switching concept for aliquoting an initial amount of liquid, typically within a range from 10 to 500 μΐ, preferably between 50 and 200 μΐ, into separate and fluidically separate aliquots, typically within a range from 1 to 100 μΐ. Embodiments of the invention are particularly suited for constant and low rotational speeds, for example within the range from 300 to 500 revolutions per minute. In embodiments of the invention, the switching concept may be applied in an apparatus suited for causing a test carrier to rotate and for directly or indirectly tempering the fluids contained therein, for example within a range between 20 °C and 100 °C. In embodiments of the invention, the apparatus used for processing the test carrier may be a commercial centrifugal thermocycler for performing polymerase chain reactions, such apparatus being distributed, e.g., by Qiagen GmbH, Hilden, Germany, under the names "Rotor-Gene 6000" or "Rotor-Gene Q". However, in embodiments the invention may be implemented with any other apparatus which has a corresponding drive means for causing the carrier to rotate, a corresponding heating means for tempering the fluid within the chambers, and a corresponding control means. Embodiments of the invention provide a possibility, for the first time, of aliquoting liquids into a plurality of fluidically separate chambers by means of a thermopneumatic effect at a constant rotational frequency. In contrast thereto, known methods have required a plurality of different rotational speeds in order to aliquot the initial amount of liquid and to subsequently transfer the individual aliquots into fluidically separated chambers. For splitting up an initial amount of liquid into several sub-volumes such that they are such that they are completely separated fluidically, known centrifugal-microfluidic systems require passive fluidic valves, at least two different rotational speeds being required for operating said valves, so that operation in standard laboratory equipment with only one rotational frequency available is not possible.

Fig. 6 schematically shows the difference between centrifugal aliquoting of an amount of liquid into several sub-volumes while using valves and without using valves. The upper part of Fig. 6 shows valve-less aliquoting wherein partial amounts of liquid are introduced into fingers which have chambers formed at their ends, without any valves between the fingers and the chambers. Following aliquoting, a liquid bridge may form between the fingers. Since after split-up, the individual aliquots are not transferred to separate chambers, there is a risk of cross contamination. The aliquots are not completely separated. The lower part of Fig. 6 shows aliquoting while using valves between the fingers and the chambers. The amount of liquid is initially split up into the individual fingers and transferred, after split-up, into separate chambers by getting past the valves. Thus, following transferal into the chambers, the aliquots are present in a manner in which they are completely separated fluidically.

In accordance with the invention, the individual liquid volumes are completely separated fluidically, so that cross contaminations between the chambers during and after aliquoting are prevented. This is achieved by subdividing the process into two steps - aliquoting into the fingers and subsequently transferring the aliquots into the chambers. The chambers may have (dry) reagents provided therein for performing reactions with the liquid volumes transferred to the chambers. Carry-over of reagents provided within the chambers cannot take place during aliquoting. Thus, the invention enables aliquoting free from carryover, since any reagents previously provided do not make contact with the fingers. Moreover, reactions which are free from cross contamination are possible within the end chambers since there is no more fluidic connection once the liquids have been transferred to the chambers.

Embodiments of the invention are suited, in particular, for fields of application in the life sciences and in medical diagnostics, wherein a sample is subdivided into several sub- volumes, the sample typically being a substance in a liquid phase. Following the subdivision, each sub-volume (aliquot) on the test carrier may be subjected to a specific test, it being possible to perform parallel determination of several diagnostically relevant parameters from a shared sample. Known centrifugal-micro fluidic systems for aliquoting an initial amount of liquid at a fixed constant rotational speed achieve no sufficient fluidic separation since the individual sub- volumes are not transferred to fluidically separate chambers, so that sub-volumes in adjacent chambers are often connected to one another via a liquid bridge. Thus, in particular with cyclic tempering, there is a risk of cross contamination; however, for medical and diagnostic applications it is crucial to avoid cross contaminations. Specifically in processes requiring increased temperatures, such as a polymerase chain reaction, for example, methods wherein cross contamination cannot be avoided are not suitable.

Embodiments of the present invention will be explained below in more detail with reference to the accompanying figures, wherein:

Fig. 1 shows a schematic top view of an embodiment of a carrier having a microfluidic structure; Figs. 2a) to 2c) show schematic representations for illustrating an embodiment of an inventive method; Figs. 3 and 4 show schematic side views of embodiments of inventive devices;

Figs. 5a and 5b show schematic representations of drive means for utilization in embodiments of the invention; and Fig. 6 shows a schematic representation for illustrating complete separation of sub-volumes of a liquid.

Fig. 1 schematically shows a top view of a section of a carrier having fluidics structures formed therein. The carrier 2 may be configured, e.g., as a body of rotation having the shape of a disc, comprising a center of rotation 4 and a central opening 6 by means of which the carrier 2 may be attached to a drive means so as to be caused to rotate, a rotational direction being indicated by an arrow ω in Fig. 1. Alternatively, the carrier may have the shape of part or of a segment of a disc. The carrier 2 comprises fluidics structures comprising an inlet chamber 10 which is fluidically connected to a distribution channel 30 via a fluid channel 20. Fingers 31a to 3 lh branch off from the distribution channel 30 and extend outward from the distribution channel 30 in a radial direction. An inlet end 30a of the distribution channel is connected to the inlet chamber via the fluid channel 20, whereas an outlet end 30b of the distribution channel leads into an overflow chamber 40. The fingers 31a to 31h define a specific volume, it being possible for the different fingers to define identical or different volumes.

Radially inner ends of the fingers 31a to 3 lh are connected to the radial exterior surface of the distribution channel. Radially outer ends of the fingers 31a to 31h are connected to unvented end chambers 33a to 33h via respective connecting channels 32a to 32h. The flow cross section of the connecting channels 32a to 32h is smaller than the respective cross sections of the fingers 31a and 3 lh and of the chambers 33a to 33h in the flow direction. The fingers 31a to 3 lh have a course which radially falls with regard to the center of rotation. The fingers need not extend along a radius, but may also have oblique or tilted positions in relation to the radius, for example. Radially falling means that the fingers have a radial component in the flow direction, i.e. that an inlet of each finger at which the finger leads into the distribution channel is located radially further inward than an outlet of each finger at which the finger is connected to the chamber.

The distribution channel 30 has an azimuthal course which radially falls from the inlet end 30a to the outlet end 30b. To compensate for this radially falling course of the distribution channel 30, the connecting channels 32a to 32h have lengths which decrease along the distribution channel 30, so that given identical lengths of the fingers 31a to 31h, the chambers 33a to 33h are each arranged at an identical radial distance from the center of rotation 4. In alternative embodiments, the end chambers may be arranged at different radial distances from the center of rotation 4.

The fluid channel 20 exhibits at least such radial components that a liquid introduced into the inlet chamber 10 may be introduced into the distribution channel 30 via the fluid channel 20 by means of centrifugal force.

The fluidics structures may be regarded as a microfluidic network.

An embodiment of a method of producing fluidically separated sub-volumes of a liquid will now be described by means of Figs. 1 and 2a) to 2c).

At the beginning of the method, a gas located within the chambers 33a to 33h, such as air, is held at an increased first temperature. For example, the gas located within the chambers 33a to 33h may be heated to the increased first temperature for this purpose. In alternative embodiments, an aerosol may be arranged within the chambers.

An initial amount of liquid is introduced into the inlet chamber 10. The liquid may be a sample having a substance in a liquid phase, for example. The carrier 2 is subject to a constant rotational speed, so that the liquid gets into the distribution channel 30 and into the individual fluidic fingers 31a to 31h from the upstream inlet chamber 10 through the fluid channel 20 due to the centrifugal force, as is shown in Fig. 2a). The liquid from the distribution channel 30 fills the fluidic finger structures 31a to 31h, the distributed liquid occluding a gas volume within the fluidic end chambers 33a to 33h. Any supernatant liquid is sheared off at the radially inner ends of the fingers 31a to 3 lh and gets into the overflow chamber 40, as is shown in Fig. 2b).

Due to the pneumatic back pressure pi within the unvented end chambers 33a to 33h at the increased first temperature, the individual portioned liquid sub-volumes (aliquots) located within the fingers cannot penetrate into the unvented end chambers 33a to 33h. However, rotation of the carrier 2 ensures that the liquids are located at a defined position at the input of the unvented end chambers 33a to 33h and/or at the input of the connecting channels 32a to 32h. Starting from the state shown in Fig. 2b), the temperature is decreased once aliquoting has taken place. For example, the temperature of the occlusion of gas within the chambers may be reduced to a lower temperature of 50 °C to 60 °C. Upon cooling of the gas within the constant volume of the end chambers 33a to 33h, the pressure within the chambers will go down to a pressure p2 < pi in accordance with the ideal gas equation. Due to the resulting underpressure, the aliquoted liquid is transferred, or drawn, from the fluidic fingers 31a to 3 lh into the end chambers 33a to 33h through the connecting channels 32a to 32h. Said transferal of the aliquoted liquid into the end chambers 33a to 33h is supported by the centrifugal force. Fig. 2c) shows the state wherein the liquid sub-volumes have been completely transferred from the fingers 3 la to 3 lh to the end chambers 33a to 33h.

In embodiments, the increased first temperature may range from 70 to 95 °C, and the lower second temperature may range from 40 to 65 °C.

The volumes of the end chambers 33a to 33h and the temperature reduction are dimensioned such that the respective complete liquid volume is transferred from the fluidic fingers 31a to 3 lh into the attached end chamber.

As was described above, the gas is heated to the increased temperature prior to aliquoting, since otherwise gas bubbles would rise within the fingers, which gas bubbles would squeeze the aliquoted liquid out of the aliquoting fingers.

Following distribution of the liquid into the fingers, the liquids within the individual fingers may possibly still be connected via a liquid film within the distribution channel. Once the portioned liquid volumes have been transferred to the chambers, the former will be completely separated from one another fluidically, however.

Thus, in embodiments of the invention, the aliquots produced are transferred to the fluidic end chambers wherein the individual aliquots are present in a manner in which they are completely separated fluidically and may be processed further in a manner which is free from cross contamination. This enables implementation of medical and diagnostic applications on the centrifugal-microfluidic systems described. Embodiments of the invention thus enable fluidic separation and transferal of an initial amount of liquid into a plurality of sub-volumes under constant rotation and in a manner which is free from cross contamination. Thus, embodiments are suited for being employed in a commercial centrifugal thermocycler.

As is shown in Fig. 3, in embodiments of the invention the carrier 2 may have the shape of a body of rotation comprising a substrate 52 and a cover 50. The substrate 52 and the cover 50 may be circular in a top view and have a central opening (6 in Fig. 1) via which the body of rotation may be mounted on a rotating part 56 of a drive means 58 via a common attachment means 54. The rotating part 56 is pivot-mounted on a stationary part 60 of the drive means 58. The drive means may be a conventional centrifuge or thermocycler, for example.

A heating means 62 is provided by means of which the carrier 2 and, thus, the gas located within the end chambers 33a to 33h may be tempered. For example, the heating means 62 may be incorporated in the rotating part 56 of the drive means 58.

In alternative embodiments, the heating means may be provided externally or may be incorporated in the body of rotation. If the heating means 62 is incorporated in the body of rotation, suitable connecting means for operating the heating means may be provided on the body of rotation and on the movable part of the drive means. In embodiments of the invention, the heating means may comprise a heater and a fan which are configured to direct a stream of air, which is tempered accordingly, onto the carrier so as to heat the entire carrier.

A control means 64 is provided for controlling the heating means 62 and the drive means 58. In embodiments of the invention, the control means controls the drive means merely by switching it on and off. Controlling in order to achieve different rotational speeds is not required since during operation, rotation at a constant rotational speed is sufficient. The control means 64 controls the heating means to adjust the temperature of the gas and/or aerosol within the end chambers 33a to 33h accordingly, i.e. to an increased first temperature while the fingers are being filled and to a reduced second temperature while the liquid is transferred to the chambers.

As is obvious to any person skilled in the art, the control means 64 may be implemented, for example, by a computing means which is programmed accordingly or by an application-specific integrated circuit.

The fluidics structures of the carrier may be formed by cavities and channels within the substrate 52. Alternatively, the fluidics structures may be formed by cavities and channels within the substrate 52 and the cover 50. In embodiments, the fluidics structures, filling openings and venting openings are formed within the substrate 52, it being possible for the cover to be unstructured. Alternatively, filling openings and venting openings may also be formed in the cover 50.

In the embodiment shown in Fig. 3, the cover 50 is arranged, in relation to the drive means 58, below the substrate 52, i.e. closer to the drive means. In alternative embodiments, the cover may be arranged above the substrate in relation to the drive means. In an alternative embodiment shown in Fig. 4, a body of rotation comprises a rotor 70 and fluidics modules 72 inserted into the rotor. The fluidics structures may be formed within the fluidics modules 72, so that in these embodiments, the carrier is formed by corresponding fluidics modules that may be inserted into a rotor. The fluidics modules 72 may have a substrate and a cover, respectively, which in turn may have the required fluidics structures formed therein. The rotor 70 and the fluidics modules 72 form a body of rotation, which in turn may be caused to rotate by the drive means 58 controlled by the control means 64.

In embodiments of the invention, the carrier and/or the cover may be formed from any suitable material, e.g. a plastic such as COC (cycloolefm copolymer), COP (cycloolefin polymer), PMMA (polymethyl methacrylate), polycarbonate, PP (polypropylene), PVC (polyvinyl chloride), or PDMS (polydimethyl siloxane), glass or the like.

As was described, a heating means 62 may be provided for tempering the entire carrier so as to result in the necessary tempering of the gas within the end chambers 33a to 33h. Possible implementations of heating means for causing local tempering which are provided in the rotating part 56 of the drive means are shown in Figs. 5a and 5b. As is shown in Fig. 5a, a heating means 62a is provided only in a small azimuthal portion of the rotatable part 56. In accordance with Fig. 5b, an annular heating means 62b is provided. The heating means 62a and 62b are provided at radial positions of the rotatable part, which correspond to the radial position of the end chambers 33a to 33h, so that gas contained within the end chambers 33a to 33h may be locally heated by the heating means 62a and 62b.

Embodiments of the invention may be performed while using centrifugal thermocyclers. A centrifugal thermocycler is understood to mean a piece of laboratory equipment for performing a polymerase chain reaction which can (cyclically) heat and cool liquid, typically between room temperature and 95°C. Sample tubes containing liquids may be located within a rotor. Depending on the design, tempering of the liquid may be performed by directing a stream of air onto it which is tempered accordingly. Such thermocyclers typically have no possibility of regulating the rotational speed implemented therein. In embodiments of the invention, the heating means may thus be implemented by a corresponding device for directing a stream of air, tempered accordingly, onto the end chambers.

Embodiments of the invention thus offer the possibility of fluidically aliquoting liquids within rotating centrifugal-microfluidic structures and subsequently transferring them to fluidically separated chambers solely by means of a change in temperature of an occlusion of gas so as to thereby rule out any detrimental cross contaminations between the aliquots produced. In embodiments of the invention, the rotational speed of the test carrier need not be reduced so as to be able to switch valves, for example. Moreover, in embodiments of the invention, it is not necessary to locally temper individual areas of a rotating test carrier. Rather, in embodiments of the invention, global tempering may be performed, which includes corresponding tempering of the gas occluded within the chambers. In embodiments, the inventive microfiuidic structure requires no additionally integrated components, structural units or surface modifications as is required in known approaches to integrating liquid retention structures. Embodiments of the invention enable integrating a test carrier having a microfiuidic structure for aliquoting within a commercial, centrifugal thermocycler. Automation of said aliquoting by using centrifugal-microfluidic systems in commercial centrifugal thermocyclers offers the possibility of clearly reducing the cost. In particular, when performing a polymerase chain reaction, as is common practice in laboratories, with a plurality of samples it has so far been required to manually produce a plurality of aliquots of an initial amount of liquid. Subsequently, a detection component specific to the respective reaction, so-called PCR primers and probes, must be supplied to each aliquot. Said manual preparation process is extremely time-consuming and susceptible to cross- contaminations. Incorporation of a rotating test carrier having a corresponding fluidic aliquoting structure into existing laboratory equipment and subsequent automated production of the completed reaction batches result in a substantial potential for savings.

Embodiments of the present invention enable, for the first time, combining centrifugal force and thermopneumatic effects for producing aliquots, which are completely separated fluidically, from an amount of liquid. In other words, embodiments of the invention provide a method of producing partial amounts of a liquid, which are completely separated fluidically, on a rotating test carrier comprising a fluidic distribution channel, several fluidic fingers branching off therefrom and having defined sub-volumes and chambers fluidically connected to the fluidic fingers, respectively. The liquid is distributed to the fluidic fingers via the distribution channel at a constant rotational speed, whereby their sub-volumes are determined and whereby a defined amount of gas becomes occluded within the attached chambers at the temperature tl . Subsequently, the temperature of the gas within the chambers is reduced to the temperature t2 < tl so as to thereby draw in the partial amounts of the liquid into the chambers at a constant rotational speed.

In embodiments of the invention, a plurality of heating and cooling steps may be employed for transferring the liquid from the fluidic fingers to the unvented chambers. In other embodiments, the liquid may be transferred from the fluidic fingers to the chambers in one portion. In embodiments of the invention, the unvented chambers may be located at a larger radial distance from the center of rotation than the fluidic fingers. In other embodiments, the unvented chambers may be radially closer to the center of rotation than the fluidic fingers.

In embodiments of the invention, the rotating test carrier is configured to be able to be operated, together with the methods described, in a commercially available centrifugal thermocycler for performing a polymerase chain reaction. The chambers may be configured such that a polymerase chain reaction may be performed therein. In embodiments of the invention, identical or different (dry) reagents may be provided within the chambers. Embodiments include a step of performing a polymerase chain reaction and/or a reverse transcription and/or an isothermal amplification of the liquid sub-volumes transferred to the chambers.

Claims

1. Method of producing fluidically separated sub-volumes of a liquid within a carrier (2) comprising a fluidics structure having a distribution channel (30) and fingers (31a-31h) branching off from the distribution channel (30) and defining a specific volume, respectively, each finger (31a-31h) being connected to an un vented chamber (33a-33h), and each finger (31a-31h) having a course, in relation to a center of rotation (4), which radially falls from the distribution channel (30) to the chamber (33a-33h) to which the finger (3 la- 3 lh) is connected, the method comprising: filling the fingers (31a-31h) with the liquid via the distribution channel (30) by rotating the carrier (2), so that the fingers (31a-31h) have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers (33a-33h) preventing the liquid from getting into the chambers (33a-33h) from the fingers (31 a-31 h); and reducing the temperature of the gas or of the aerosol within the chambers (33a-33h), so that an underpressure will result within the chambers (33a-33h) which causes the liquid volumes to be drawn into the chambers (33a-33h) from the fingers (31a-31h).

2. Method as claimed in claim 1, wherein a rotational speed of the carrier (2) is kept constant while the fingers (31 a-31 h) are being filled and while the temperature of the gas or of the aerosol within the chambers (33a-33h) is reduced.

3. Method as claimed in claims 1 or 2, wherein the distribution channel (30) comprises an azimuthal course which radially falls from an inlet end (30a) to an outlet end (30b) thereof, said filling of the fingers (31a-31h) comprising introducing the liquid into the inlet end, shearing off the liquid at a radially inner end of each finger (31 a- 3 lh), and emptying the distribution channel (30) into an overflow chamber (40) fluidically connected to the outlet end (30b) of the distribution channel (30).

4. Method as claimed in any of claims 1 to 3, wherein prior to the filling step the gas or aerosol within the chambers (33a-33h) is heated to a first temperature, the temperature being reduced, after the filling, to a second temperature lower than the first temperature, so that the liquid volumes are drawn into the chambers (33 a-33 h) from the fingers (3 la-3 lh).

5. Method as claimed in claim 4, wherein the first temperature ranges from 70 to 95°C and wherein the second temperature ranges from 40 to 65°C.

6. Method as claimed in any of claims 1 to 5, wherein due to the reduction of the temperature, the entire liquid volumes are transferred from the fingers (31a-31h) into the chambers (33a-33h).

7. Method as claimed in any of claims 1 to 5, wherein a plurality of heating and cooling steps are used for transferring the liquid volumes from the fingers (31 a-31 h) into the chambers (33a-33h).

8. Method as claimed in any of claims 1 to 7, wherein the chambers (33a-33h) are radially spaced further apart from the center of rotation (4) than are the fingers (31 a- 31h).

9. Method as claimed in any of claims 1 to 7, wherein the chambers (33a-33h) are radially closer to the center of rotation (4) than the fingers (3 la-3 lh).

10. Method as claimed in any of claims 1 to 9, further comprising a step of performing a polymerase chain reaction and/or a reverse transcription and/or an isothermal amplification in the liquid volumes within the chambers (33a-33h).

11. Method as claimed in any of claims 1 to 10, wherein reducing the temperature of the gas comprises reducing the temperature of the carrier (2).

12. Device for producing fluidically separated sub-volumes of a liquid, comprising: a carrier (2) comprising a fluidics structure having a distribution channel (30) and fingers (31a-31h) branching off from the distribution channel (30) and defining a specific volume, respectively, each finger (31a-31h) being connected to an unvented chamber (33a-33h), and each finger having a course, in relation to a center of rotation (4), which radially falls from the distribution channel (30) to the chamber (33a-33h) to which the finger (31a-31h) is connected; a drive means (58) configured to cause the carrier (2) to rotate so as to rotate the carrier (2) around the center of rotation (4) so as to fill the fingers (31 a-31 h) with the liquid via the distribution channel (30), so that the fingers (31a-31h) have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers (33a-33h) preventing the liquid from getting into the chambers (33 a- 33h) from the fingers (31a-31h) while the fingers (31 a-31 h) are being filled with the liquid; a heating means (62) configured to temper a fluid within the chambers (33a-33h); and a control means (64) configured to control the heating means (62) to heat a gas or aerosol within the chambers (33a-33h) to a first temperature prior to the fingers (31 a-31 h) being filled with the liquid, and control the heating means (62) to reduce, following filling of the fingers (31a-31h), the temperature of the gas or aerosol within the chambers (33a-33h) to a second temperature, which is lower than the first temperature, so that an underpressure will result within the chambers (33a-33h) which causes the liquid volumes to be drawn into the chambers (33a-33h) from the fingers (31a-31h). filling the fingers (31a-31h) with the liquid via the distribution channel (30) by rotating the carrier (2), so that the fingers (31a-31h) have portioned liquid volumes contained therein, gas volumes or aerosol volumes occluded within the chambers (33a-33h) preventing the liquid from getting into the chambers (33a-33h) from the fingers (31a-31h); and reducing the temperature of the gas or of the aerosol within the chambers (33a-33h), so that an underpressure will result within the chambers (33a-33h) which causes the liquid volumes to be drawn into the chambers (33a-33h) from the fingers (31a-31h).

Device as claimed in claim 12, wherein the control means (64) is configured to

control the drive means (58) to keep constant a rotational speed of the carrier (2) while the fingers (31a-31h) are being filled and while the temperature of the gas or aerosol within the chambers (33a-33h) is being reduced.

Device as claimed in claims 12 or 13, wherein the distribution channel (30) comprises an azimuthal course which radially falls from an inlet end (30a) to an outlet end (30b) thereof, the inlet end (30a) of the distribution channel (30) being connected to an upstream fluidic structure (10), and the outlet end (30b) of the distribution channel (30) being fluidically connected to an overflow chamber (40), the fingers (31a-31h) extending radially from the distribution channel (30) at mutually spaced apart positions, said filling of the fingers (31a-31h) comprising introducing the liquid into the inlet end of the distribution channel (30), shearing off the liquid at a radially inner end of each finger (3 la-3 lh), and emptying the distribution channel (30) into an overflow chamber (40) fluidically connected to the outlet end (30b) of the distribution channel (30).

15. Device as claimed in any of claims 12 to 14, wherein the chambers (33a-33h) are end chambers comprising no air vent beside the fluidic connection to the respective finger (3 la-31h).

16. Device as claimed in any of claims 12 to 15, wherein the chambers (33a-33h) are radially spaced further apart from the center of rotation (4) than are the fingers (31 a- 31h).

17. Device as claimed in any of claims 12 to 16, wherein the chambers (33a-33h) are radially closer to the center of rotation (4) than the fingers (3 la-3 lh).

18. Device as claimed in any of claims 12 to 17, wherein the chambers (33a-33h) are arranged at an identical radial distance from the center of rotation (4), the heating means (62) being arranged on the drive means (58) or the carrier (2) at the same radial distance from the center of rotation (4).

19. Device as claimed in any of claims 12 to 18, wherein the fingers (31 a-31 h) are fluidically connected to the associated chambers (33a-33h) via a connecting channel

(32a-32h), a flow cross section of the connecting channels (32a-32h) being smaller than a cross section of the fingers (31a-31h) and of the chambers (33 a-33 h) transverse to the flow direction.

Device as claimed in claim 19 when dependent on claim 13, wherein the length of the connecting channels (32a-32h) decreases along the distribution channel (30), so that the chambers (33a-33h) are arranged at an identical radial distance from the center of rotation (4).

Device as claimed in any of claims 12 to 20, wherein the heating means is configured to temper the carrier (2).