Patent Description:
The document <CIT> describes a cartridge with a waste zone, such that the cartridge together with the waste is discarded after process completion. <CIT> shows a droplet actuator with discrete and continuous flow sections separated by a barrier and <CIT> concerns a self-containing disposable cartridge.

It is a task of the current invention to provide a cartridge that allows for a precise and versatile processing of microfluidic droplets.

This task is solved by a cartridge with the features of claim <NUM>. Further embodiments of the cartridge, an electrowetting sample processing system with such a cartridge, as well as a method for operating such a cartridge or system are defined by the features of further claims.

A cartridge according to the invention, namely a cartridge for use in an electrowetting sample processing system, comprises one or more inlet ports for introducing an input liquid into an internal gap of the cartridge. The gap comprises at least one hydrophobic surface for enabling an electrowetting induced movement of multiple microfluidic droplets separated from the input liquid. The cartridge further comprises at least one outlet port that is operably connected to the inlet port for providing a liquid flow through the cartridge, if a liquid driving force, in particular an electrowetting force or a pressure force, is applied to at least a part of the input liquid. The cartridge comprises a first part with the inlet port and a second part attached to the first part, such that the gap is formed between the first part and the second part, the second part comprising an electrode support element or a flexible film.

The cartridge comprises a first part with the inlet port and a second part attached to the first part, such that the gap is formed between the first part and the second part.

The second part comprises an electrode support element or a flexible film.

In a further embodiment, the first part comprises a rigid body and/or the second part comprises or is a polymer film. In particular, the second part is attached to a peripheral side structure of the first part.

In a further embodiment, the gap is defined by a spacer that is arranged between the first part and the second part and/or by the shape of at least one of the two parts of the cartridge, in particular by a flexible part or a rigid part of the cartridge.

In a further embodiment, one or more of the following comprise an outlet port: the first part, the second part, the spacer, the peripheral side structure of the first part.

In a further embodiment, the cartridge is configured to provide the flow through the cartridge as a continuous flow and/or to substantially maintain a volume equilibrium in the cartridge. The continuous flow may include periods of unbalanced pressure, for example an under-pressure or an over-pressure, which may result from differences of flow between the input flow and the output flow, i.e. differences in the pumping characteristic. In one example, the maximum length of the period of unbalanced pressure is <NUM> second, in particular <NUM> seconds.

In a further embodiment, the cartridge comprises a plurality of electrodes, in particular an electrode array, for applying an electrowetting force to the microfluidic droplets.

In an embodiment, the second part of the cartridge, in particular the electrode support element or the flexible film or the membrane, is reversibly attachable to the electrodes of the electrowetting sample processing system.

In a further embodiment, at least two of the electrodes are connected to an electrical interface, in particular to an electrical connector or contact field.

In a further embodiment, the cartridge comprises the inlet port as a single inlet port.

In a further embodiment, the cartridge is configured as a disposable cartridge and/or as cartridge that is removably attachable to an electrowetting sample processing system.

In a further embodiment, the input liquid comprises a carrier liquid and/or an electrowetting filler liquid, further in particular a silicone oil.

In a further embodiment, the input liquid comprises a processing liquid that comprises at least one of:.

In a further embodiment, the cartridge comprises at least one liquid removal element, in particular a line removal and/or a removal zone, that is operably connected to the outlet port.

In a further embodiment, the cartridge comprises a pressure compensation outlet and/or an air ventilation outlet for providing a fluid output arranged separate from the outlet port, in particular gas exhaust.

The features of the above-mentioned embodiments of the cartridge can be used in any combination, unless they contradict each other.

An electrowetting sample processing system according to the present invention, in particular a biological sample processing system, comprises a cartridge according to anyone of the above-mentioned embodiments.

An electrowetting sample processing system according to the invention comprises a cartridge (<NUM>) with an internal gap and one or more inlet ports for introducing an input liquid into the internal gap. The gap comprises at least one hydrophobic surface for enabling an electrowetting induced movement of multiple microfluidic droplets separated from the input liquid. The internal gap further comprises at least one outlet port that is in operable connection with the inlet port for providing a liquid flow through the internal gap, if a liquid driving force, in particular an electrowetting force or a pressure force, is applied to at least a part of the input liquid. The cartridge comprises a first part with the inlet port and a second part attached to the first part, such that the gap is formed between the first part and the second part.

In an embodiment, the electrowetting sample processing system comprises a plurality of electrodes for applying an electrowetting force to the microfluidic droplets, in particular an electrode array, further in particular a two-dimensional electrode array.

In an embodiment, at least two of the electrodes are connected to an electrical interface, in particular to an electrical connector or contact field.

In an embodiment, the electrowetting sample processing system comprises a cartridge, which is reversibly attachable to the electrodes of the electrowetting sample processing system, wherein in particular the cartridge comprises a electrode support element or a flexible second part, further in particular a flexible film or the membrane.

In an embodiment, the electrowetting sample processing system or the cartridge comprises a processing zone, which is configured for processing samples, in particular for processing biological sample, and/or which is operably connected to the delivery zone.

In an embodiment, the processing zone is configured for processing least one of:.

In an embodiment, the processing zone is configured for processing a PCR (Polymerase chain reaction) process and/or a hybridization.

In an embodiment, the electrowetting sample processing system comprises a liquid feeder operably connected to the inlet port by a tube, in particular a flexible tube, for feeding the input liquid to the inlet port.

In an embodiment, the liquid feeder is configured to provide the input liquid as sequential feed and/or alternating feed of a processing liquid and a carrier liquid.

In an embodiment, the liquid feeder is configured to provide the input liquid as feed of at least two processing liquids of different compositions separated by an carrier liquid.

In an embodiment, the liquid feeder comprises a T-shaped junction and/or a multi-port valve for providing the input liquid.

In an embodiment, the liquid feeder comprises a bypass that is controllable for flushing a tube of the feeder and/or for removing an access liquid from a feeding liquid and to providing the remaining part of the feeding liquid as the input liquid.

In an embodiment, the liquid feeder comprises a control element, in particular a pump and/or a multi-port valve, for introducing the input liquid into the internal gap and/or for removing an output liquid from the internal gap.

In an embodiment, the liquid feeder is configured to operate independently and/or asynchronously from the operation of electrodes used for electrowetting.

In an embodiment, the input liquid comprises at least one of:.

In an embodiment, the electrowetting sample processing system comprises a reagent detector for indicating the presence of processing liquid in the input liquid and/or for monitoring the amount of processing liquid in the input liquid, in particular in relation to a predetermined value.

The features of the above-mentioned embodiments of the electrowetting sample processing system can be used in any combination, unless they contradict each other.

The invention further concerns a method for operating the cartridge according to the invention or the sample processing system according to the invention.

The invention further concerns a method for operating a cartridge according to the invention or a sample processing system according to the invention that comprises an internal gap, which comprises one or more inlet ports, an outlet port and at least one hydrophobic surface enabling an electrowetting induced movement of microfluidic droplets separated from the input liquid. The method comprises:.

In an embodiment, the driving force is provided by a plurality of electrodes, in particular by an electrode array, further in particular by a two-dimensional electrode array.

In an embodiment, the step of providing the flow through the internal gap as a substantially continuous flow and/or maintaining a volume equilibrium.

In an embodiment, the method comprises inducing a movement of multiple microfluidic droplets by operating a plurality of electrodes, in particular an electrode array (<NUM>), for applying the electrowetting force to the microfluidic droplets.

In an embodiment, the input liquid comprises a carrier liquid and/or an electrowetting filler liquid, in particular a silicone oil. In a further embodiment, the input liquid comprises a processing liquid that comprises at least one of:.

The features of the above-mentioned embodiments of the method can be used in any combination, unless they contradict each other.

Embodiments of the current invention are described in more detail in the following with reference to the figures. These are for illustrative purposes only and are not to be construed as limiting.

The <FIG> shows an overview over an electrowetting sample processing system exemplary shown as digital microfluidics system <NUM> that is equipped with a central control unit <NUM> and a base unit <NUM>, with four cartridge accommodation sites <NUM> that each comprise an electrode array <NUM>, and a cover plate <NUM>. The digital microfluidics system <NUM> is configured for manipulating samples in liquid droplets <NUM> within cartridges designed as disposable cartridges <NUM>. This digital microfluidics system <NUM> also comprises four board accommodation sites <NUM> for receiving an electrode board <NUM>.

The digital microfluidics system <NUM> comprises a base unit <NUM> with at least one cartridge accommodation site <NUM> that is configured for taking up a disposable cartridge <NUM>. The digital microfluidics system <NUM> can be a standalone and immobile unit, on which a number of operators are working with cartridges <NUM> that they bring along. The digital microfluidics system <NUM> thus may comprise a number of cartridge accommodation sites <NUM> and a number of electrode arrays <NUM> at least some of which are located on electrode boards <NUM>.

It may be preferred to integrate the digital microfluidics system <NUM> into a liquid handling workstation or into a Freedom EVO® robotic workstation, so that a pipetting robot can be utilized to transfer liquid portions and/or sample containing liquids to and from the cartridges <NUM>.

Alternatively, the system <NUM> can be can be configured as a hand-held unit which only comprises and is able to work with a low number, e.g. a single disposable cartridge <NUM>. Every person of skill will understand that intermediate solutions that are situated in-between the two extremes just mentioned will also operate and work within the gist of the present invention.

According to the present invention, the digital microfluidics system <NUM> also comprises at least one board accommodation site <NUM> for taking up an electrode board <NUM> which comprises an electrode array <NUM> that substantially extends in a first plane and that comprises a number of electrodes <NUM>. Such an electrode board <NUM> preferably is located at each one of said cartridge accommodation sites <NUM> of the base unit <NUM>. Preferably each electrode array <NUM> is supported by a bottom substrate <NUM>. It is noted that the expressions "electrode array", "electrode layout", and "printed circuit board (PCB)" are utilized herein as synonyms.

The digital microfluidics system <NUM> may also comprise at least one cover plate <NUM> with a top substrate; though providing of such cover plates <NUM> is particularly preferred, at least some of the cover plates may be dispensed with or may be re-placed by an alternative cover for holding a disposable cartridge <NUM> in place inside the base unit of the microfluidics system <NUM>. Thus, at least one cover plate <NUM> may be located at one of said cartridge accommodation site <NUM>. The cover plate <NUM> and the bottom substrate <NUM> with the electrode array <NUM> or PCB define a space or cartridge accommodation site <NUM> respectively. In a first variant (see the two cartridge accommodation sites <NUM> in the middle of the base unit <NUM>, the cartridge accommodation sites <NUM> are configured for receiving a slidingly inserted disposable cartridge <NUM> that is movable in a direction substantially parallel with respect to the electrode array <NUM> of the respective cartridge accommodating site <NUM>. Such front- or top-loading can be supported by a drawing-in automatism that, following a partial insertion of a disposable cartridge <NUM>, transports the cartridge <NUM> to its final destination within the cartridge accommodation site <NUM>, where the cartridge <NUM> is precisely seated. Preferably, these cartridge accommodation sites <NUM> do not comprise a movable cover plate <NUM>. After carrying out all intended manipulations to the samples in liquid droplets, the used cartridges <NUM> can be ejected by the drawingin automatism and transported to an analysis station or discarded.

In a second variant (see the two cartridge accommodation sites <NUM> on the right and left of the base unit <NUM>), the cartridge accommodation sites <NUM> comprise a cover plate <NUM> that is configured to be movable with respect to the electrode array <NUM> of the respective cartridge accommodating site <NUM>. The cover plate <NUM> preferably is configured to be movable about one or more hinges <NUM> and/or in a direction that is substantially normal to the electrode array <NUM>.

Similar to the possibilities for inserting a disposable cartridge <NUM> into a cartridge accommodation site <NUM>, possibilities for inserting the electrode board <NUM> into a board accommodation site <NUM> comprise the following alternatives:.

In <FIG>, there is drawn only one electrode board <NUM> that slidingly can be inserted by front loading below the second cartridge accommodation site <NUM> (as counted from the left). All possible places for locating a board accommodation site <NUM> are indicated and pointed to by dashed arrows.

The digital microfluidics system <NUM> also comprises a central control unit <NUM> for controlling the selection of the individual electrodes <NUM> of said at least one electrode array <NUM> and for providing these electrodes <NUM> with individual voltage pulses for manipulating liquid droplets within said cartridges <NUM> by electrowetting. As partly indicated in <FIG>, every electrode <NUM> is operatively connected to the central control unit <NUM> and therefore can be independently or commonly addressed by this central control unit <NUM>, which also comprises the appropriate sources for creating and providing the necessary electrical potentials in a way known in the art.

The at least one cover plate <NUM> preferably comprises an electrically conductive material that extends in a second plane and substantially parallel to the electrode array <NUM> of the cartridge accommodation site <NUM> the at least one cover plate <NUM> is assigned to. It is particularly preferred that this electrically conductive material of the cover plate <NUM> is configured to be not connected to a source of an electrical ground potential. The cover plate <NUM> can be configured to be movable in any arbitrary direction and no electrical contacts have to be taken in into consideration when selecting a particularly preferred movement of the cover plate <NUM>. Thus, the cover plate <NUM> may be configured to be also movable in a direction substantially parallel to the electrode array <NUM> and for carrying out a linear, circular or any arbitrary movement with respect to the respective electrode array <NUM> of the base unit <NUM>.

The <FIG> shows a section view of one exemplary cartridge accommodation site <NUM> with the disposable cartridge <NUM> according to <FIG> accommodated therein. The disposable cartridge <NUM> comprises a bottom layer <NUM> as a second part of the cartridge <NUM>, a top layer <NUM> as a first part of the cartridge <NUM>, and a spacer <NUM> that defines a gap between the bottom and top layers <NUM>,<NUM> for manipulating samples in liquid droplets <NUM> in this gap <NUM>.

The cover plate <NUM> is mechanically connected with the base unit <NUM> of the digital microfluidics system <NUM> via a hinge <NUM>; thus, the cover plate <NUM> can swing open and a disposable cartridge <NUM> can be placed on the cartridge accommodation site <NUM> via top-entry loading (see <FIG>). An electrically conductive material <NUM> of the cover plate <NUM> is configured as a thin metal plate or metal foil that is attached to the top substrate <NUM>. Alternatively, the electrically conductive material <NUM> of the cover plate <NUM> is configured as a metal layer that is deposited onto the top substrate <NUM>. Such deposition of the conductive material <NUM> may be carried out by chemical or physical vapor deposition techniques as they are known per se.

The cover plate <NUM> is configured to apply a force to a disposable cartridge <NUM> that is accommodated at the cartridge accommodation site <NUM> of the base unit <NUM>. This force urges the disposable cartridge <NUM> against the electrode array <NUM> in order to position the bottom layer <NUM> of the cartridge as close as possible to the surface of the electrode array <NUM>. This force also urges the disposable cartridge <NUM> into the perfect position on the electrode array <NUM> with respect to an optional piercing facility <NUM> of the cover plate <NUM>. This piercing facility <NUM> is configured for introducing sample droplets into the gap <NUM> of the cartridge <NUM>. The piercing facility <NUM> is configured as a through hole <NUM> that leads across the entire cover plate <NUM> and that enables a piercing pipette tip <NUM> to be pushed through and pierce the top layer <NUM> of the cartridge <NUM>. The piercing pipette tip <NUM> may be a part of a handheld pipette (not shown) or of a pipetting robot (not shown).

In the case shown in <FIG>, the electrode array <NUM> is covered by a dielectric layer <NUM>. The electrode array <NUM> is fixed to a bottom substrate <NUM>, this combination is also called PCB, and every individual electrode <NUM> is electrically and operationally connected with the central control unit <NUM> (only three connections of the ten electrodes <NUM> are drawn here). Alternatively, the electrodes may be commonly connected to an electrical interface, in particular to an electrical connector or an electrical contact field - which in turn is then electrically connected to a control unit <NUM>. In one example, the bottom substrate <NUM> or the PCB that contains the electrode array <NUM> or the electrodes <NUM> has an electrical connector, which connects to a relay PCB, which is connected to a control PCB, wherein the control PCB is part of the central control unit <NUM>.

The electrode array <NUM> is located on an immovably fixed bottom substrate <NUM>. The digital microfluidics system <NUM> is configured for manipulating samples in liquid droplets <NUM> within disposable cartridges <NUM> that contain a gap <NUM>. Accordingly, the samples in liquid droplets <NUM> are manipulated in the gap <NUM> of the disposable cartridge <NUM>. The disposable cartridge <NUM> comprises the bottom layer <NUM>, the top layer <NUM>, and the spacer <NUM> that defines the gap <NUM> between the bottom and top layers <NUM>,<NUM> for manipulating samples in liquid droplets <NUM> in this gap <NUM>. The bottom layer <NUM> and the top layer <NUM> comprise a hydrophobic surface <NUM> that is exposed to the gap <NUM> of the cartridge <NUM>. The bottom layer <NUM> and the top layer <NUM> of the cartridge <NUM> are entirely hydrophobic films or at least comprise a hydrophobic surface that is exposed to the gap <NUM> of the cartridge <NUM>. It is clear from this <FIG>, that the cartridge <NUM> does not have a conductive layer. The spacer <NUM> of the cartridge <NUM> may optionally be configured as a body that includes compartments <NUM> for reagents needed in an assay that is applied to the sample droplets in the gap <NUM> (dotted lines).

<FIG> shows a section view of a further exemplary cartridge accommodation site according to <FIG> with a cartridge <NUM>, wherein - in contrast to <FIG> - the cartridge <NUM> comprises an electrode array <NUM>' of individual electrodes <NUM>.

Further the cartridge <NUM> comprises an upper part <NUM>, a spacer <NUM>, a hydrophobic layer <NUM>'', a support element <NUM>' for the electrode array <NUM>', an optional through hole <NUM>, a liquid input port <NUM>' and electrically conductive material. The upper part <NUM> and the spacer <NUM> may be provided as separate parts or in form of a single piece. The hydrophobic layer <NUM>'', the electrode array <NUM>' and the support element <NUM>' form the lower part of the cartridge. The electrode array <NUM>' is arranged between the hydrophobic layer <NUM>'' and the support element <NUM>' and the gap is formed between the upper part <NUM> and the hydrophobic layer <NUM>''. Further, the hydrophobic layer <NUM>'' is attached to a peripheral side structure of the upper part <NUM> resp. to the spacer <NUM>. The support element <NUM>' further comprises electrical connectors <NUM>', which are connected via multiple electrical wires to the electrode array <NUM>'. In turn, the electrical connectors <NUM>' provide for a connection to a central control unit <NUM> such that the electrical connectors <NUM>' implement an electrical interface between cartridge <NUM> and the digital microfluidics system <NUM>. The electrical interface can also be implemented by a contact field, i.e. a plurality of electrically conductive, mutually insulated contact areas.

<FIG> shows section view of one cartridge accommodation site <NUM> with a disposable cartridge <NUM> according to a further embodiment accommodated therein. Again, the electrodes <NUM> are arranged on and fixed to the bottom substrate <NUM>. Again, the disposable cartridge <NUM> comprises a bottom layer <NUM>' and a top layer <NUM>. Attached to the disposable cartridge <NUM> is a spacer <NUM> that defines a gap <NUM> between the bottom and top layer <NUM>, <NUM> for manipulating samples in liquid droplets <NUM> in this gap <NUM>. In this embodiment, the bottom layer is a flexible bottom layer, for example a membrane <NUM>', for example with a hydrophobic surface <NUM>. For example, the membrane <NUM>' is an <NUM> to <NUM> thick polypropylene film. The bottom layer <NUM>' is arranged between the top layer <NUM> and the spacer <NUM>.

Preferably, the flexible bottom layer <NUM> is reversibly attached to the electrodes <NUM> in an electrowetting sample processing system <NUM>. The spacer <NUM> may be a part of the cartridge <NUM> or a part of the electrowetting sample processing system <NUM>. In one example, the spacer <NUM> comprises stainless steel, aluminum, hard plastic, in particular COP or ceramic. The spacer <NUM> may be designed to define the height of the gap <NUM>. The spacer <NUM> may additionally serve as a gasket for sealing the gap <NUM>.

Preferred dimensions and materials are pointed to in table <NUM>. These indications of materials and dimensions serve as preferred examples without limiting the scope of the present invention.

An inlet port <NUM>' for introducing a liquid <NUM>,<NUM> into the gap <NUM> is provided in the top layer <NUM> of the cartridge <NUM>. In addition, an outlet port <NUM> is provided for removing liquid from the gap <NUM> of the cartridge <NUM>. The outlet port <NUM> is arranged in this case also in the top layer <NUM> of the cartridge <NUM>. Preferably, the top layer <NUM> comprises a rigid body when the inlet port <NUM>' and/or an outlet port <NUM> are arranged within the top layer <NUM>, to provide a certain stability to the ports <NUM>',<NUM>. Stability is desired to ensure a sufficiently tight connection of tubes <NUM> of an outer liquid circuit to the ports, so that the liquid does not leak at the connection between the port(s) <NUM>',<NUM> of the cartridge <NUM> and the tubes <NUM>.

Preferably, the inlet port <NUM>' and the outlet port <NUM> are operably connected. By this, a liquid flow is provided through the cartridge <NUM> if a liquid driving force is applied to at least a part of the input liquid <NUM>. A liquid driving force may be a pressure force applied to at least a part of the input liquid <NUM> and/or an electrowetting force for example applied to at least a part of the input liquid <NUM> when it has been moved into the gap <NUM> of the cartridge <NUM>.

In addition, a vacuum supply line <NUM> is exemplarily shown in <FIG>. By means of a vacuum supply line <NUM>, the bottom layer <NUM>' may be attached tightly to the surface of the electrodes <NUM>.

<FIG> shows a section view of an exemplary cartridge accommodation site with a disposable cartridge according to a further embodiment accommodated therein. Also, this cartridge <NUM> comprises a flexible bottom layer <NUM>' which is arranged on the electrodes <NUM>. In this embodiment, the top layer <NUM> comprises peripheral side structures <NUM> which define the gap <NUM>. For defining the gap <NUM>, the peripheral side structures <NUM> are preferably rigid structures. The bottom layer <NUM>' is attached to the peripheral side structures <NUM>. Thus, in this embodiment shown, the gap <NUM> is defined by the shape and dimensions of the top layer <NUM>. It is possible to combine the use of spacer <NUM> with the shape of the top layer <NUM> or with a shape of the bottom layer <NUM> for defining the gap <NUM>.

An inlet port <NUM>' is provided again in the top layer <NUM> of the cartridge <NUM>. By means of the inlet port <NUM>', an input liquid <NUM>,<NUM> can be introduced into the cartridge <NUM>. An inlet port <NUM>' may also be arranged in the side structures of the cartridge <NUM>, for example in a spacer <NUM> or in peripheral side structures <NUM> of the top layer <NUM>, depending on the chosen structure of the cartridge <NUM>. In one example, the inlet port <NUM>' is located on the bottom layer and enters the spacer <NUM> and - after a <NUM> degree turn - enters into the side of the cartridge <NUM>. In another example the gap spacer that enables the liquid connection is located in the middle or a center part of the cartridge <NUM>.

In the embodiment of <FIG>, two outlet ports <NUM> are provided in the cartridge <NUM>: one outlet port <NUM> is arranged in the top layer <NUM>, and one outlet port <NUM> is arranged in a peripheral side structure <NUM> of the top layer <NUM>. A certain degree of rigidity of the top layer <NUM> and its side structure <NUM> ensures a dimensionally stable gap <NUM> and also dimensionally stable ports <NUM>',<NUM>.

The number of outlet ports <NUM> provided by a cartridge <NUM> may depend on the application for which the cartridge <NUM> is designed. According to the invention, at least one inlet port <NUM>' and at least one outlet port <NUM> are provided, to enable a liquid flow throughout the cartridge <NUM>. In another example multiple inlet ports <NUM>' are used, which in particular provide input for different reagents or different classes of reagents, for example at least one bulk reagent via a first inlet port and at least one stoichiometric reagent via a second inlet port. In another example, the multiple inlet ports <NUM>' are individually connected to a control element, in particular to a multi-port valve <NUM> and/or to a pump, to an individual input syringe pump <NUM> and/or an individual T-shaped junction <NUM>.

In <FIG>, the inlet port <NUM>' and one of the outlet ports <NUM> comprise a seal <NUM>. Such a seal may help to achieve a tight connection between the cartridge <NUM> and an "outer" liquid circuit or tubing system.

<FIG> shows a schematic view of an exemplary embodiment of an inlet port <NUM>'. A first connecting sleeve <NUM> is arranged at the top of the top layer <NUM>. The first connecting sleeve <NUM> is formed integrally with the top layer <NUM>. The first connecting sleeve <NUM> comprises a centering cone <NUM> at its inside, wherein the centering cone <NUM> faces away from the top layer <NUM> and widens with an increased distance to the top layer <NUM>. A supply channel <NUM> is formed by a tube <NUM> and a second connecting sleeve <NUM> with a centering cone <NUM> at its inside. During the assembly, the tube <NUM> is centered by the centering cone <NUM> of the first connecting sleeve <NUM>. When completely inserted, the tube <NUM> forms a tight connection with the first connecting sleeve <NUM> and the seal <NUM> provided by the centering sleeve <NUM>. The centering cone <NUM> of the second connecting sleeve <NUM> faces the top layer <NUM> and widens with a reduced distance to the top layer <NUM>. The inside of the second connecting sleeve <NUM> is bigger than the outside of the first connecting sleeve <NUM>. During the assembly, the second connecting sleeve <NUM> is centered by the outside of the first connecting sleeve <NUM>. The free inner space in the inlet port <NUM>' forms the inlet channel <NUM>".

Alternatively, an inlet port <NUM>' and/or an outlet port <NUM> may be a simple passage opening into which a tube of an outer liquid circuit may be mounted. Preferably, the passage opening comprises a seal <NUM> for a tight connection.

<FIG> shows a schematic overview over an exemplary embodiment of an electrowetting sample processing system <NUM> with a cartridge <NUM>, an inlet port <NUM>' and operably connected outlet ports. This particular embodiment comprises three distinct outlet ports <NUM>, with which liquid <NUM>,<NUM>, may be removed from the cartridge <NUM>.

By the operational connection of the inlet port <NUM>' and the outlet ports <NUM>, a liquid flow through the cartridge <NUM> is provided, if a liquid driving force is applied to at least a part of the input liquid <NUM>. The input liquid <NUM> may comprise a carrier liquid <NUM> and/or a processing liquid <NUM>.

A particular suitable carrier liquid <NUM> is an electrowetting filler liquid, for example a silicone oil. In an embodiment, an additional carrier liquid, for example a silicone oil may be used. An electrowetting filler liquid may be used for filling the gap <NUM>, while a carrier liquid may be used to a liquid which segments droplets in the droplet generator. In one embodiment, the electrowetting filler liquid and the carrier liquid may be the same liquid. In a further embodiment, the electrowetting filler liquid is a different liquid, for example a different oil than the liquid used as a carrier liquid.

A processing liquid <NUM> can be any kind of liquid or liquid composition which is used for example in assay reactions or for analysis purposes or other applications carried out in the cartridge <NUM>. Such a processing liquid <NUM> may be for example buffers, reaction liquids which comprise reactants required for a defined application, sample liquids which comprise a sample to be analyzed, diluent liquids, elution liquids, etc. Samples are for example DNA (Desoxyribonucleic acid), RNA (Ribonucleic Acid), derivatives thereof, proteins, cells, or other biologically or biochemically derived molecules or combinations thereof. The processing liquid <NUM> may in an embodiment comprise magnetic beads, to which for example one or more samples are bound.

Applications which may be carried out using a cartridge <NUM> and an electrowetting sample processing system <NUM> are for example at least one of chemical reactions, washing processes, heating processes, polymerase chain reaction (PCR) processes, hybridization processes, mixing processes, dilution processes, and NGS (next-generation sequencing) library prep assays.

The liquid flow through the cartridge <NUM> is realized by removing an amount of liquid as an output liquid <NUM> from the cartridge <NUM> via the outlet port <NUM> which is equivalent to the amount of input liquid <NUM> introduced into the cartridge <NUM>. This operational linkage allows for example to maintain the level of liquid in the cartridge <NUM>. Additionally, droplets of a determined volume may be generated outside the gap <NUM> of the cartridge <NUM>, which allows a more precise volume control. This further allows storage of input liquids outside the cartridge <NUM>, which removes requirements on the design of the gap <NUM> and the electrode array concerning for example the temperature of delicate liquids or volume requirements for bulk amounts of liquid. By removing such requirements from the cartridge <NUM>, the design of the cartridge is more flexible, which allows the integration of more and/or more complex applications within one cartridge <NUM>. The output liquid <NUM> may comprise at least a part of carrier liquid <NUM>, processing liquid <NUM>, sample containing liquid or any combinations thereof. The output liquid <NUM> may be treated as a waste liquid, which is discarded, or may be used for further processing.

Preferably, the delivery of the input liquid <NUM> through a liquid inlet port <NUM>' into the gap <NUM> for an electrowetting induced movement is synchronized with the electrowetting control, to ensure a proper hand-off of droplets <NUM>. However, other processes outside the cartridge <NUM> required beforehand of the delivery may be carried out independently from the operations of electrodes <NUM> used for electrowetting. Though some processes may be synchronized, others may be carried out asynchronously from the operation of the electrodes <NUM>, as described in the following.

Possible liquid operations in connection with the liquid inlet port <NUM>' and the outlet port(s) <NUM> are shown exemplarily in <FIG>: the cartridge <NUM> comprises one liquid inlet port <NUM>' and three outlet ports <NUM> for liquids. Via the liquid inlet port <NUM>', input liquid <NUM> may be introduced into the internal gap <NUM> of the cartridge <NUM>. The input liquid <NUM> is provided from respective storage tubes <NUM>. A multi-port valve <NUM> is in this case operably connected to the storage tubes <NUM>, and additionally to the liquid inlet port <NUM>', directly or via further elements such as a T-shaped junction. The connection is realized here by a tube <NUM> or a tube system comprising multiple tubes <NUM> that are in fluid connection to each other, so that the input liquid <NUM> is fed into the inlet port <NUM>' from the storage place into the cartridge <NUM>. Preferably, the tube <NUM> is a flexible tube.

The liquid feeder <NUM> comprises a multi-port valve <NUM> which allows for the movement of liquids <NUM>,<NUM> within the tubes <NUM> for introducing into the internal gap <NUM> of the cartridge <NUM>, wherein in particular, the liquid feeder <NUM> comprises a liquid selector valve, for example a multiport valve, that works with an input syringe pump <NUM> for providing the liquid movement within the tubes. Because the inlet port <NUM>' and the outlet ports <NUM> are connected operationally, liquid may additionally be removed from the gap <NUM> of the cartridge <NUM> by means of a further pump or the waste pump <NUM>. Liquid <NUM>,<NUM> which has been removed from the cartridge <NUM> via the outlet port <NUM> is transported via tubes <NUM> for example to a waste collecting place. Tubes <NUM> which are involved in the removal of waste liquids are shown in light grey, the direction of liquid movement within the tubes <NUM> is indicated by dashed arrows.

The movement of liquids within the tubes <NUM> may be supported by providing one or more additional pumps <NUM>,<NUM> operably connected to the tube(s) <NUM>, e.g. via a bypass <NUM>. In the embodiment of the electrowetting sample processing system <NUM> shown in <FIG>, the removal of waste liquids is supported by the right one of the syringe pumps, which acts as an outlet syringe pump <NUM> whereas the left input syringe pump <NUM> particularly supports the movement of the input liquid <NUM>. Waste liquids may then be collected in distinct waste collection containers, for example.

Waste management may further be supported by providing a removal line <NUM> within the cartridge <NUM>. Such a removal line <NUM> is typically formed by a specific array of electrodes which guide input liquid <NUM>,<NUM> immediately from the inlet port <NUM>' to an outlet port <NUM>. This allows the immediate removal for example of wash fluid from the inlet port <NUM>'. As shown in <FIG>, a removal zone <NUM>, for example adjacent to the outlet port <NUM>, may additionally be provided for collecting waste droplets prior their removal from the cartridge <NUM>.

The liquid feeder <NUM> of the embodiment of the electrowetting sample processing system <NUM> shown in <FIG> further comprises a T-shaped junction <NUM> for providing a predetermined volume of input liquid <NUM>,<NUM> to the liquid inlet port <NUM>'. Alternatively or in addition, the liquid feeder <NUM> may comprises a multi-port valve <NUM> as shown for example in <FIG> and <FIG>.

At the T-shaped junction <NUM> the cross-flow is used for generating droplets of a defined volume. In particular, an carrier liquid <NUM> is pumped towards the T-shaped junction <NUM> by the left syringe pump <NUM> in alternating coordination with a processing liquid <NUM> from the direction of the liquid feeder <NUM>, which is pumped into the T-shaped junction <NUM> in a cross-flow direction. By alternating flows of processing liquid <NUM> and carrier liquid <NUM>, droplets of processing liquid <NUM> of controlled volume, separated by the carrier liquid <NUM>, are generated.

Two different processing liquids <NUM> of a defined volume are generated by the T-shaped junction <NUM>, indicated by white and black droplets, and are transported towards the liquid inlet port <NUM>'. The distinct volumes of processing liquid <NUM> are separated by specific volumes of carrier liquid <NUM>, so that the processing liquids <NUM> may be fed to the inlet port <NUM>' alternatingly with the carrier liquid <NUM>. Depending on the flow of the carrier liquid <NUM> towards the T-shaped junction <NUM> and the feeding of different processing liquids <NUM> into the tube towards that junction <NUM>, it is also possible to provide the input liquid <NUM> as a sequential feed of a single type of processing liquid <NUM> or a sequential feed of two or more processing liquids <NUM>, which differ in their composition.

The liquid feeder <NUM> comprises a multi-port valve <NUM> and four separate storage tubes <NUM>, which are in fluid connection with the multi-port valve <NUM>, wherein in each storage tube <NUM> stores a different processing liquid <NUM>. In another example, up to <NUM>, in particular up to <NUM>, separate storage tubes <NUM> with different processing liquids are in fluid connection with the multi-port valve <NUM>. Preferably, at least one storage tube <NUM> is provided and connected to the multi-port valve <NUM>, however, multiple storage tubes <NUM> are preferred to provide sufficient storage space for different processing liquids <NUM>.

In addition to the processing liquid <NUM>, the carrier liquid <NUM> may be in fluid connection to the liquid feeder as well, as indicated for the supply tube <NUM> on the rightmost side of the multi-port valve <NUM>.

In the present embodiment, the carrier liquid <NUM> comprises the same material composition as the electrowetting filler liquid, namely a silicon oil. In another example, the carrier liquid <NUM> and the electrowetting filler liquid <NUM> may be different, e.g. silicon oil with different viscosities.

The liquid feeder <NUM> additionally comprises a bypass <NUM> for removing access liquid from liquids which are to be provided as input liquid <NUM>. Other liquids such as liquids for flushing one or more tubes <NUM> of the liquid feeder <NUM> may also be removed from the tubular connection with the inlet port <NUM>' by using the bypass <NUM>.

In this example, the cartridge <NUM> further comprises an air ventilation outlet <NUM> for providing a fluid output that is arranged separate from the outlet port. Here, the air ventilation outlet <NUM> serves as gas exhaust, in another example, the pressure cartridge comprises a compensation outlet such as a liquid overflow.

In this embodiment, the electrowetting sample processing system <NUM> comprises a reagent detector <NUM> for indicating the presence of reagent liquid in the input liquid, for example by detecting at least one characteristic of the reagent liquid, in particular an optical characteristic such as transmissivity (resp. absorbance) or refraction index or an electrical characteristic such as resistance (resp. conductivity) or capacity.

<FIG> shows a schematic overview over another exemplary embodiment of an electrowetting sample processing system <NUM>. The overall elements correspond to the elements as described in <FIG>. The embodiment of <FIG> uses as a mechanism for storing and providing processing liquids a reagent carousel <NUM> in combination with one or more pipettes <NUM> for aspirating liquids from the storage place in the reagent carousel <NUM>. The reagent carousel <NUM> preferably comprises a rotation mechanism for positioning a desired storage place in relation to the aspiration pipette <NUM>. The aspiration pipette <NUM> is connected to the tube <NUM> for feeding the liquid inlet port <NUM>' and may be configured to work automatically. For automatic aspiration of processing liquid <NUM>, the pipette <NUM> may be configured to be movable at least along a Z-axis of a Cartesian coordinate system.

<FIG> shows exemplarily in a schematic view how at a T-shaped junction <NUM> a droplet of a processing liquid <NUM> is formed from a bulk droplet of that liquid, followed by a volume of carrier liquid <NUM>. Flow directions of liquid <NUM>,<NUM> within the tube <NUM> are indicated by dashed arrows. A bulk droplet of a processing liquid <NUM> is moved for example by the input syringe pump <NUM> and the multi-port valve <NUM> of the liquid feeder <NUM> forwards into the T-shaped junction <NUM>, while the flow of the carrier liquid <NUM> is stopped (see situation in A and B). When the flow of the carrier liquid <NUM> is started, this flow shears of an initial droplet of processing liquid <NUM> from the leading volume of the bulk droplet where the two fluid paths cross each other. A fluid path cross point as present in the T-shaped junction <NUM> serves as a shear location (see situation C). The generated droplet may be pumped within the tube <NUM> towards the liquid inlet port <NUM>', or into an alternative tube <NUM>, for example into the bypass <NUM>. By repeating the alternating flow of processing liquid <NUM> and carrier liquid <NUM> into the T-shaped junction <NUM>, a train of droplets of processing liquid <NUM> and intermediate liquid parts of carrier liquid <NUM> is generated, which may be fed to the liquid inlet port <NUM>' of the cartridge.

<FIG> shows a schematic view of T-shaped junctions <NUM> where droplets <NUM> of uniform volumes are generated. For this, each droplet is sheared at both ends by using an alternating flow of two different liquids <NUM>,<NUM> at the T-shaped junction <NUM>. The initial droplet which is generated from a bulk droplet at the shear location is sacrificed, so that the uncertainty of the position of the leading edge of the bulk droplet, which causes an uncertainty of the exact volume of the initial droplet generated, is removed. Again, the flow directions of liquid <NUM>,<NUM> within the tube <NUM> are indicated by dashed arrows.

As shown in situation A, a bulk droplet of processing liquid <NUM> is pumped into the T-shaped junction <NUM>, and an initial volume of liquid <NUM> crosses the shear location. By starting the flow of the carrier liquid <NUM> towards the shear point in the T-shaped junction <NUM>, an initial droplet of processing liquid <NUM> is sheared of the bulk droplet at the shear point. The initial droplet of processing liquid <NUM> is discarded and may be pumped, for example by means of separate pump like a syringe pump <NUM>, into a waste path for removal. The new leading edge of the bulk droplet of processing liquid <NUM> is now at a defined position within the T-shaped junction <NUM> (situation B), so that the following droplets may be generated under controlled conditions. The residual bulk droplet (situation C) is discarded as well to ensure that the last droplet is created by shearing on both ends.

<FIG> shows different views of a multi-port valve <NUM> which is configured for droplet generation independently from electrowetting processes in the cartridge <NUM>. To the multi-port valve <NUM>, a number tubes <NUM> are connected for providing the carrier liquid <NUM> and/or different processing liquids <NUM> into the valve. A dashed arrow indicates in each situation A to E the direction of liquid flow in the tube <NUM> after droplet generation. Liquid flow may be controlled by a suction action of a syringe pump <NUM>, for example, by the application of pressure using a syringe pump <NUM>, or by other means for controlling the flow of liquids in a tubular system. For generating a droplet <NUM> of a processing liquid <NUM>, which is embedded within a carrier liquid <NUM>, the supply tubes <NUM> for the liquids are filled, and the carrier liquid <NUM>, for example a silicone oil, is guided into the valve (situation A). Upon switching the valve <NUM> from oil to a processing liquid <NUM> (situation B), followed by switching back to the oil supply tube <NUM> (situation C), an alternating flow of carrier fluid <NUM> and processing fluid <NUM> is formed.

An example of moving liquid towards a waste removal processing is shown for the situations D and E. This process may be used when for example an air gap is located within the processing liquid <NUM> and the carrier liquid <NUM>. Using a syringe pump, reagents are pumped into the valve <NUM> with the air gap between the silicone oil and the processing liquid (situation D). Then, the valve is switched to a tube <NUM> which is connected with the waste removal system, and the air is pumped to the waste removal processing place.

<FIG> shows a schematic view of an exemplary electrowetting sample processing system <NUM> comprising a multi-port valve <NUM> and a T-shaped junction <NUM> for droplet generation and another embodiment of tube arrangement and combination of elements for guiding liquids <NUM>,<NUM> into and from the cartridge <NUM>. General details may be taken from the <FIG>.

Claim 1:
A cartridge (<NUM>) for use in an electrowetting sample processing system, the cartridge comprising one or more inlet ports (<NUM>') for introducing an input liquid (<NUM>) into an internal gap (<NUM>) of the cartridge (<NUM>), which comprises at least one hydrophobic surface (<NUM>) for enabling an electrowetting induced movement of multiple microfluidic droplets (<NUM>) separated from the input liquid (<NUM>),
wherein the cartridge (<NUM>) further comprises at least one outlet port (<NUM>) for removing liquid from the internal gap (<NUM>), wherein the at least one outlet port (<NUM>) is operably connected to the inlet port (<NUM>') for providing a continuous liquid flow through the internal gap (<NUM>), if a liquid driving force is applied to at least a part of the input liquid,
characterized in that the cartridge (<NUM>) comprises a first part (<NUM>) with the inlet port (<NUM>') and a second part (<NUM>) attached to the first part (<NUM>), such that the gap (<NUM>) is formed between the first part (<NUM>) and the second part (<NUM>), the second part (<NUM>) comprising an electrode support element (<NUM>') or a flexible film (<NUM>').