Patent Description:
This technical field generally relates to the control and manipulation of liquids in a small volume, usually in the micro- or nanoscale format. In digital microfluidics, a defined voltage is applied to electrodes of an electrode array, so that individual droplets are addressed (electrowetting). For a general overview of the electrowetting method, please see <NPL>, and <NPL>. Briefly, electrowetting refers to a method to move liquid droplets using arrays of microelectrodes, preferably covered by a hydrophobic layer. By applying a defined voltage to electrodes of the electrode array, a change of the surface tension of the liquid droplet, which is present on the addressed electrodes, is induced. This results in a remarkable change of the contact angle of the droplet on the addressed electrode, hence in a movement of the droplet. For such electrowetting procedures, two principle ways to arrange the electrodes are known: using one single surface with an electrode array for inducing the movement of droplets or adding a second surface that is opposite a similar electrode array and that provides at least one ground electrode. A major advantage of the electrowetting technology is that only a small volume of liquid is required, e.g. a single droplet. Thus, liquid processing can be carried out within considerably shorter time. Furthermore the control of the liquid movement can be completely under electronic control resulting in automated processing of samples.

Automated liquid handling systems are generally well known in the art. An example is the Freedom EVO® robotic workstation from the present applicant (Tecan Schweiz AG, Seestrasse <NUM>, CH-<NUM> Männedorf, Switzerland). These automated systems are larger systems that are not designed to be portable and typically require larger volumes of liquids (microliter to milliliter) to process.

A device for liquid droplet manipulation by electrowetting using one single surface with an electrode array (a monoplanar arrangement of electrodes) is known from the <CIT>. All electrodes are placed on a surface of a carrier substrate, lowered into the substrate, or covered by a non-wettable surface. A voltage source is connected to the electrodes. The droplet is moved by applying a voltage to subsequent electrodes, thus guiding the movement of the liquid droplet above the electrodes according to the sequence of voltage application to the electrodes.

An electrowetting device for microscale control of liquid droplet movements, using an electrode array with an opposing surface with at least one ground electrode is known from <CIT> (a biplanar arrangement of electrodes). Each surface of this device may comprise a plurality of electrodes. The two opposing arrays form a gap. The surfaces of the electrode arrays directed towards the gap are preferably covered by an electrically insulating, hydrophobic layer. The liquid droplet is positioned in the gap and moved within a non-polar filler fluid by consecutively applying a plurality of electric fields to a plurality of electrodes positioned on the opposite sites of the gap.

Containers with a polymer film for manipulating samples in liquid droplets thereon are known from <CIT>: a biological sample processing system comprises a container for large volume processing and a flat polymer film with a lower surface and a hydrophobic upper surface. The flat polymer film is kept at a distance to a base side of the container by protrusions. This distance defines at least one gap when the container is positioned on the film. A substrate supporting at least one electrode array is also disclosed as well as a control unit for the liquid droplet manipulation instrument. The container and the film are reversibly attached to the liquid droplet manipulation instrument either separately or stably connected to each other in the form of a disposable cartridge. The system enables displacement of at least one liquid droplet from the at least one well through a channel of the container onto the hydrophobic upper surface of the flat polymer film and above the at least one electrode array. The liquid droplet manipulation instrument is accomplished to control a guided movement of said liquid droplet on the hydrophobic upper surface of the flat polymer film by electrowetting and to process there the biological sample.

The use of such an electrowetting device for manipulating liquid droplets in the context of the processing of biological samples is also known from the international patent application published as <CIT>. There, it is disclosed that a droplet actuator typically includes a bottom substrate with the control electrodes (electrowetting electrodes) insulated by a dielectric, a conductive top substrate, and a hydrophobic coating on the bottom and top substrates. The cartridge may include a ground electrode, which may be replaced by a hydrophobic layer, and an opening for loading samples into the gap of the cartridge. Interface material (e.g. a liquid, glue or grease) may provide adhesion of the cartridge to the electrode array.

Disposable cartridges for microfluidic processing and analysis in an automated system for carrying out molecular diagnostic analysis are disclosed in <CIT> (see <CIT> for an English translation). The cartridge is configured as a flat chamber device (with about the size of a check card) and can be inserted into the system. A sample can be pipetted into the cartridge through a port and into processing channels.

Droplet actuator structures are known from the international patent application <CIT>. This document particularly refers to various wiring configurations for electrode arrays of droplet actuators, and additionally discloses a two-layered embodiment of such a droplet actuator which comprises a first substrate with a reference electrode array separated by a gap from a second substrate comprising control electrodes. The two substrates are arranged in parallel, thereby forming the gap. The height of the gap may be established by spacer. A hydrophobic coating is in each case disposed on the surfaces which face the gap. The first and second substrate may take the form of a cartridge, eventually comprising the electrode array.

From <CIT>, a digital microfluidics system for manipulating samples in liquid droplets within disposable cartridges is known. The disposable cartridge comprises a bottom layer, a top layer, and a gap between the bottom and top layers. The digital microfluidics system comprises a base unit with at least one cartridge accommodation site that is configured for taking up a disposable cartridge, at least one electrode array comprising a number of individual electrodes and being supported by a bottom substrate, and a central control unit for controlling selection of the individual electrodes of said at least one electrode array and for providing these electrodes with individual voltage pulses for manipulating liquid droplets within said cartridges by electrowetting.

A disposable cartridge having a body with at least one compartment configured to hold therein processing liquids, reagents or samples (a disposable cartridge for microfluidics system) is known from <CIT>, incorporated herein by explicit reference. The disposable cartridge further comprises a bottom layer with a first hydrophobic surface that is configured as a working film for manipulating samples in liquid droplets thereon. Further comprised is a top layer with a second hydro-phobic surface that is attached to a lower surface of the body. The bottom layer is configured as a flexible film that is sealingly attached to the top layer along a circumference of the flexible bottom layer. The disposable cartridge thus being devoid of a spacer that is located between the flexible bottom layer and the top layer for defining a particular distance between said first hydrophobic surface and said second hydrophobic surface.

A digital microfluidics system configured for substantially removing or suspending magnetically responsive beads from or in liquid portions or droplets (magnetic conduits in microfluidics) is known from <CIT>. Said digital microfluidics system comprises a number or array of individual electrodes attached to a first substrate, wherein a first hydrophobic surface is located on said individual electrodes. Further comprised is a central control unit in operative contact with said individual electrodes. In the first substrate of the microfluidics system and below said individual electrodes there is located at least one magnetic conduit that is configured to be backed by a backing magnet, said at least one magnetic conduit being located in close proximity to individual electrodes. In this system, magnetically responsive beads are removed from droplets on a working surface in digital microfluidics by means of the integration of the magnetic conduit into the PCB of a digital microfluidics device.

Electrowetting is a versatile approach to automate complex assays for life sciences and diagnostic point-of-care markets. The integration of an electrowetting platform with a robotic liquid handler enables the delivery of samples and reagents inside the fluidic chamber (gap) of a disposable cartridge used in electrowetting in a digital microfluidics system whenever needed and with a large range of volumes possible (<NUM>-<NUM>µl). However, such approach is not optimal for point-of-care diagnostic markets for the reasons below:
One problem relies on reagents and samples containment to prevent contamination. Traditional liquid handlers rely on a centralized high-performance positive-displacement pump for the aspiration and the dispensing of liquids either via a syringe (e.g. Tecan Cavro® Centris Pump) or a piston (e.g. Tecan Cavro® Air Displacement Pipettor). Certain assays are highly sensitive to minute contamination: for example, single-molecule assays or assays requiring a large number of cycles during PCR amplification. Minute contamination can come from variable sources such as the creation of an aerosol during liquid dispensing or during the ejection of disposable tips, imperfect washing of fixed tips, contamination of system fluid in a syringe pump, or open samples/reagents vials that need to be accessible to the liquid handler. These assays often require processes to be separated onto different instruments or even in different laboratory rooms.

Another problem relies on instrument footprint, weight and cost. Traditional liquid handlers have a robotic arm to move the liquid handler between the reagent vials and the cartridge. This configuration requires a non-negligible amount of space and prevents the instrument from being compact. The weight and cost of <NUM>-axis motors, metallic supports, driving belts, and other mechanical components are not really compatible with the concept of a point-of-care instrument. Also, a centralized high-performance syringe pump represents an unnecessary cost to the instrument because the precision of the reagent dispensing is controlled by electrowetting and only approximate volumes of reagents need to be injected into the electrowetting cartridge.

Therefore, interfacing with a robotic liquid handler is a problem in the state of the art.

Lyophilization is a dehydration process commonly used for the preservation of perishable reagents during transportation and long-term storage at room temperature. Lyophilized reagents could be stored inside the fluidic chamber of a disposable cartridge used in electrowetting and re-solubilized by a buffer solution whenever needed. However, the lack of physical barriers inside the fluidic chamber can result in lyophilized beads shifting within the disposable cartridge especially during transportation and ending up being at the wrong location or, even worse, contaminating unwanted areas of the disposable cartridge. The implementation of containment features within the disposable cartridge used in electrowetting would involve complex manufacturing processes and thus a less cost-effective consumable. Additionally, not all reagents can be lyophilized (e.g. alcohols) and would need to be loaded by the user whenever needed thus removing the convenience of a true walkaway solution.

Therefore, embedding lyophilized reagents into the fluidics chamber of a disposable cartridge used in electrowetting is a problem in the state of the art.

It is further a problem in the state of the art that certain reagents (e.g. enzymes, fluorophores, HPP substrate) need to be kept under specific conditions (low temperature, protected from light) prior to their usage to remain fully functional or to prevent the formation of unwanted byproduct. A further problem relies on that non-polar reagents (ethanol, isopropanol) cannot be exposed to the filler fluid for long periods of time especially at high temperatures in order to prevent their slow dissolution. Further, fluid capacity (≤ <NUM>µl) for storing individual reagents inside the cartridge is limited by the height of the fluidic chamber and may not be sufficient if repeated operations are needed (e.g. wash buffer). Reagent containment is of paramount importance when dealing with assays highly sensitive to minute contamination. Complex and tedious assays often require a large number of reagents (≥ <NUM>) which, if loaded manually, could lead to improper loading and thus assay failure.

It is an object of the present disclosure to suggest a cover for use in a digital micro-fluidics system resolving problems in the state of the art.

This object is achieved in that it is proposed that the cover introduced at the beginning further comprises on one side the second hydrophobic surface and on another side at least one micro-container interface for safe introducing into and/or withdrawing of liquids from the gap. Moreover, the at least one micro-container interface comprises at least one cone, the inner surface thereof being formed such to provide a sealing form fit contact with an outer surface of an inserted micro-container nozzle, by which a liquid is transferrable through a fluidic access hole formed into the cover and interconnecting each cone and the gap.

It is another object of the present disclosure to suggest a micro-container resolving problems in the state of the art.

This object is achieved by a micro-container for use in a digital microfluidics system for manipulating samples in liquid portions or droplets. The micro-container comprises a tube, a nozzle with an aperture, and a piston sealingly guided inside the tube for dispensing or aspirating liquid via the nozzle of the micro-container. The outer surface of the nozzle of the micro-container is formed such to provide a sealing form fit contact with an inner surface of a cone comprised by a micro-container interface of a cover as herein disclosed.

It is another object of the present disclosure to suggest a means adapted to proper and easily accommodate at least one micro-container.

This object is achieved by a manifold comprising at least one micro-container receptacle adapted to accommodate a micro-container as herein disclosed.

It is another object of the present disclosure to suggest a method of introducing into and/or withdrawing liquid from a gap of a digital microfluidics system.

The invention concerns a method according to claim <NUM> for introducing liquid into a gap of a digital microfluidics system.

In a first aspect, this object is achieved by a method of introducing liquid into a gap of a digital microfluidics system for manipulating samples in liquid portions or droplets; the digital microfluidics system comprising a first substrate and a central control unit, wherein said first substrate comprises an array of electrodes, and wherein said central control unit is in operative connection to said electrodes for controlling the selection of individual electrodes thereof and for providing a number of said electrodes with voltage for manipulating liquid portions or droplets by electrowetting; in said digital microfluidics system, a working gap with a gap height is located parallel to the array of electrodes and in-between first and second hydrophobic surfaces; the two hydrophobic surfaces facing each other at least during operation of the digital microfluidics system. The method comprises the steps of:.

In a second aspect, this object is achieved by a method of withdrawing liquid from a gap of a digital microfluidics system for manipulating samples in liquid portions or droplets, the digital microfluidics system comprising a first substrate and a central control unit, wherein said first substrate comprises an array of electrodes, and wherein said central control unit is in operative connection to said electrodes for controlling the selection of individual electrodes thereof and for providing a number of said electrodes with voltage for manipulating liquid portions or droplets by electrowetting; in said digital microfluidics system, a working gap with a gap height is located parallel to the array of electrodes and in-between first and second hydrophobic surfaces; the two hydrophobic surfaces facing each other at least during operation of the digital microfluidics system. The method comprises the steps of:.

Additional and inventive features and preferred embodiments and variants of the cover, the micro-container, the manifold and the methods derive from the respective dependent claims.

Advantages of the present invention comprise:.

Advantageously, such pre-packaged reagents would not require special handling (e.g. temperature) during transportation and storage.

Aspects, embodiments and examples are described with the help of the attached schematic drawings that show selected and exemplary embodiments of the present disclosure without narrowing the scope. It is shown in:.

<FIG> shows a cross sectional view of a first embodiment of a cover <NUM> with an introduced micro-container <NUM> in a partial view. In particular, <FIG> illustrates a disposable cartridge <NUM> for use in a digital microfluidics system <NUM> for manipulating samples in liquid portions or droplets, only a small part of the disposable cartridge <NUM> being visualized though. The digital microfluidics system <NUM> comprises a first substrate <NUM> and a central control unit <NUM> for controlling the selection of individual electrodes <NUM> of an electrode array <NUM> comprised by the first substrate <NUM>. The first substrate <NUM> is comprised by the digital microfluidics system <NUM>. The central control unit <NUM> is configured for providing a number of said electrodes <NUM> with voltage or rather individual voltage pulses for manipulating liquid portions or droplets by electrowetting. The disposable cartridge <NUM> comprises a first hydrophobic surface <NUM> and the cover <NUM>, the bottom of which is provided with a second hydrophobic surface <NUM>. In the following, the first hydrophobic layer <NUM> can be referred as hydrophobic working layer.

According to the first embodiment, it is to be noted that the first hydrophobic surface <NUM> is comprised by the disposable cartridge <NUM>. The second hydrophobic surface <NUM> is comprised by the cover <NUM>, the latter being part of a disposable cartridge <NUM>. In any case however, the two hydrophobic surfaces <NUM>,<NUM> are facing each other at least during operation of the digital microfluidics system <NUM>.

In the first embodiment as shown in <FIG>, the first and second hydrophobic surfaces <NUM>,<NUM> are both comprised by the disposable cartridge <NUM> configured to be positioned on the array of electrodes <NUM> of the first substrate <NUM>. Both hydrophobic surfaces <NUM>,<NUM> are facing each other at least during operation of the digital microfluidics system <NUM> and are separated or separable in essentially parallel planes by a gap <NUM> with a gap height. The cover <NUM> comprises on one side the second hydrophobic surface <NUM> and on another side at least one micro-container interface <NUM> for safe introducing into and/or withdrawing of liquids from the gap <NUM>. Said at least one micro-container interface <NUM> comprises at least one cone <NUM>, wherein the inner surface thereof being formed such to provide a sealing form fit contact with an outer surface of an inserted micro-container nozzle <NUM>.

In the first embodiment as shown in <FIG>, the cover <NUM> as well as the first and second hydrophobic surfaces <NUM>,<NUM> are comprised by the disposable cartridge <NUM>, which is configured to be positioned on the array of electrodes <NUM> of the first substrate <NUM>. The disposable cartridge <NUM> comprises a working film <NUM> with the first hydrophobic surface <NUM> and the cover <NUM> comprises the second hydrophobic surface <NUM>. Said second hydrophobic surface <NUM> is separated or separable from said first hydrophobic surface <NUM> by said gap <NUM>. The working film <NUM>, if placed on the digital microfluidics system <NUM>, comprises a backside that touches an uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>. A liquid portion is transferrable via said micro-container nozzle <NUM> and through a fluidic access hole <NUM> formed into the cover <NUM> and inter-connecting each cone <NUM> and the gap <NUM>. The diameter D of the aperture of the micro-container nozzle <NUM> can equal the diameter of the fluidic access hole <NUM> and preferably measures ≤ <NUM> or ≤ <NUM>.

It is preferred that the digital microfluidics system <NUM> comprises at least one clamping means <NUM> for establishing good mechanical contact between the disposable cartridge <NUM> and the uppermost surface of the substrate <NUM>. In doing so, the cover <NUM> is clamped or rather held in place on the uppermost surface of the first substrate <NUM> by means of the least one clamping means <NUM> of the digital microfluidics system <NUM>. It is further preferred that at least a part of the at least one clamping means <NUM> of the digital microfluidics system <NUM> is configured to press onto a free area of the cover <NUM> of the disposable cartridge <NUM> that is properly placed on the substrate <NUM> of the digital microfluidics system <NUM>.

According to the embodiment as depicted in <FIG>, a method of introducing liquid <NUM> into the gap <NUM> of the digital microfluidics system <NUM> for manipulating samples in liquid portions or droplets is provided, wherein the digital microfluidics system <NUM> comprises a first substrate <NUM> and a central control unit <NUM>, wherein said first substrate <NUM> comprises an array of electrodes <NUM>, and wherein said central control unit <NUM> is in operative connection to said electrodes for controlling the selection of individual electrodes <NUM> thereof and for providing a number of said electrodes with voltage for manipulating liquid portions or droplets by electrowetting. In said digital microfluidics system <NUM>, a working gap <NUM> with a gap height is located parallel to the array of electrodes <NUM> and in-between the first and second hydrophobic surfaces <NUM>,<NUM>; wherein the two hydrophobic surfaces <NUM>,<NUM> are facing each other at least during operation of the digital microfluidics system <NUM>. The method comprises a first step of placing the cover <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM>, wherein the cover <NUM> comprises on one side the second hydrophobic surface <NUM> and on another side the at least one micro-container interface <NUM>, wherein said at least one micro-container interface <NUM> comprises at least one cone <NUM> with an inner surface and at least one fluidic access hole <NUM> formed into the cover <NUM> and inter-connecting each cone <NUM> and the gap <NUM>. A second step comprises providing an essentially uniform height of the gap <NUM> between said first and second hydrophobic surfaces <NUM>,<NUM>. A third step comprises inserting the nozzle <NUM> of the at least one micro-container <NUM> filled with the liquid <NUM> into the at least one cone <NUM> of the micro-container interface <NUM> of the cover <NUM>. A forth step comprises creating a sealing form fit contact between the inner surface of the cone <NUM> of the micro-container interface <NUM> and the outer surface of the nozzle <NUM> of the inserted micro-container <NUM>. A fifth step comprises dispensing the liquid <NUM> from the at least one micro-container <NUM> into the gap <NUM> via the fluidic access hole <NUM> formed in the cover <NUM>.

The method can further comprise the step of clamping the placed cover <NUM> on the first substrate <NUM> by means of the at least one clamping means <NUM> of the digital microfluidics system <NUM>.

According to the first embodiment, in a first aspect as depicted on the left side of <FIG>, the cover <NUM> of the disposable cartridge <NUM> is configured rigid or flexible. At least one spacer <NUM> is attached to the cover <NUM> such to sealingly enclose the gap <NUM>. Said spacer <NUM> defines the height of the gap <NUM> between the first and second hydro-phobic surfaces <NUM>,<NUM> of the disposable cartridge <NUM>. Further, the spacer <NUM> permanently separates the first and second hydrophobic surfaces <NUM>,<NUM> from each other. Preferably the spacer <NUM> is located close to the outer circumference of the disposable cartridge <NUM>; however, additional and intermediately located spacers (not shown) may enable the utilization of a less rigid and/or thinner cover <NUM> with its first hydro-phobic surface <NUM>. While not shown, the cover <NUM> of the disposable cartridge <NUM> can be configured flexible.

In the first embodiment, in the first aspect as shown on the left side of <FIG>, a method of introducing liquid into the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein the disposable cartridge <NUM> comprises a working film <NUM> with the first hydro-phobic surface <NUM> and the cover <NUM> comprising the second hydrophobic surface <NUM>, wherein the cover <NUM> of the disposable cartridge <NUM> is configured rigid or flexible, at least one spacer <NUM> being attached to the cover <NUM>, the second hydro-phobic surface <NUM> being separated from said first hydrophobic surface <NUM> by said gap <NUM>, wherein said working film <NUM> comprises a backside that is configured to touch an uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>. This method further comprises a sixth step of sealingly enclosing the gap <NUM> with the spacer <NUM>. The method further comprises a seventh step of defining with the spacer <NUM> the height of the gap <NUM> between the first and second hydrophobic surfaces <NUM>,<NUM> of the disposable cartridge <NUM>, and permanently separating the first and second hydrophobic surfaces <NUM>,<NUM>. Further, the method comprises an eighth step of positioning the disposable cartridge <NUM> on the array of electrodes <NUM> of the first substrate <NUM> of the digital microfluidics system <NUM>.

According to the first embodiment, in a second aspect as shown on the right side of <FIG>, the cover <NUM> of the cartridge <NUM> is rigid and the working film <NUM> of the cartridge <NUM> is flexible. In other words, the working film <NUM> of the disposable cartridge <NUM> is configured as a flexible sheet that spreads on the uppermost surface of the substrate <NUM> of the digital microfluidics system <NUM>. For doing so, the digital microfluidics system <NUM> preferably comprises a vacuum source (not shown) for establishing an underpressure in an evacuation space between the uppermost surface of the substrate <NUM> and the backside of the working film <NUM> of the disposable cartridge <NUM>. Further, at least one gasket <NUM> can be attached to the cover <NUM> and outside of the gap <NUM> for separating said first and second hydrophobic surfaces <NUM>,<NUM> when creating the underpressure between the backside of the working film <NUM> and the uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>. In a non-shown alternative, the gasket <NUM> can be attached to the uppermost surface of the substrate <NUM>. Moreover, providing a rigid gasket <NUM> as a loose insert is also possible. However, in this second aspect of the embodiment, the gasket <NUM> is outside of the gap <NUM> and also on the outside of the working film <NUM>. The gasket <NUM> seals an evacuation space against the environment when the underpressure is established inside the evacuation space using the vacuum source of the digital microfluidics system <NUM>. The flat spreading of the working film <NUM> provides an essentially uniform height of the gap <NUM>, wherein this gap height is defined by the height of the gasket <NUM>. In this second aspect, the disposable cartridge <NUM> is devoid of a spacer (refer to first aspect) that would need to be located inside the gap <NUM> between the working film <NUM> and the second hydrophobic surface <NUM> of the rigid cover <NUM>.

In the first embodiment, in the second aspect as shown on the right side of <FIG>, a method of introducing liquid into the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein the cover <NUM> is comprised by a disposable cartridge <NUM>, the disposable cartridge <NUM> comprising a working film <NUM> with the first hydrophobic surface <NUM> and the cover <NUM> comprises the second hydrophobic surface <NUM>, the cover <NUM> of the disposable cartridge <NUM> is configured rigid and the working film <NUM> of the disposable cartridge <NUM> is configured flexible; wherein at least one gasket <NUM> being attached to the cover <NUM> and outside of the gap <NUM> for separating said first and second hydrophobic surfaces <NUM>,<NUM>. The method further comprises a sixth step of positioning the disposable cartridge <NUM> on the array of electrodes <NUM> of the first substrate <NUM> of the digital microfluidics system <NUM>. The method further comprises a seventh step of creating an underpressure between the backside of the working film <NUM> and the uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>. Further, the method comprises an eighth step of spreading the working film <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM> and establish the gap height.

In the scope of the present invention, a "sample" is defined in its broadest sense. A "sample" may be present in or introduced into e.g. an aqueous liquid portion or droplet for example as a biopolymer, e.g. such as nucleic acid or protein; a biomonomer, e.g. such as nucleic base or amino acid; as ions in buffers; as solvents; and as reagents. These "samples" are listed for illustration only but not for limiting interpretation of the expression "sample".

As mentioned above, according to the first embodiment as shown in <FIG>, the cover <NUM> comprises on one side the second hydrophobic surface <NUM> and on the other side at least one micro-container interface <NUM> (only one being shown here) for safe introducing into and/or withdrawing of liquids from the gap <NUM>. Such introducing or withdrawing preferably is carried out by the nozzle <NUM> of the micro-container <NUM> via the fluidic access hole <NUM> formed into the cover <NUM>. Said at least one micro-container interface <NUM> comprises the cone <NUM>, wherein the inner surface thereof formed such to provide a sealing form fit contact with an outer surface of the inserted nozzle <NUM> of the micro-container <NUM>, by which nozzle <NUM> liquid is transferred through the fluidic access hole <NUM> formed into the cover <NUM> and interconnecting each cone <NUM> and the gap <NUM>. This cone <NUM> also is configured to prevent the nozzle <NUM> from touching the first hydrophobic surface <NUM>. The micro-container <NUM> further comprises a tube <NUM> formed integrally with the nozzle <NUM>. The tube <NUM> receives a piston <NUM> allowing movement thereof in an axially direction. The micro-container <NUM> is filled with a liquid <NUM>. The micro-container can be adapted to transfer a sample to the digital micro-fluidics system, said sample preferably is selected from body fluids, e.g. from the group comprising blood, saliva, urine, and feces.

<FIG> shows a cross sectional view of a second embodiment of a cover <NUM> with an introduced micro-container <NUM> in a partial view. In this Figure, same components as shown in <FIG> are given similar reference signs. In particular, the second embodiment depicted in <FIG> differs from the first embodiment as depicted in <FIG> in that the first hydrophobic surface <NUM> is not comprised by a cartridge Like the embodiment shown in <FIG>, the second hydrophobic surface <NUM> is comprised by the cover <NUM>. The cover <NUM> is configured as a rigid plate and to be accommodated on the first substrate <NUM>. The cover <NUM> comprises the spacer <NUM> for separating said first and second hydrophobic surfaces <NUM>,<NUM> when accommodating the cover <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM>. Alternatively, the spacer <NUM> can be comprised by the first substrate <NUM>. As a further option, the spacer <NUM> can be provided separately; in this option, the spacer <NUM> is affixed to neither the cover <NUM> nor the first substrate <NUM>. This separate spacer <NUM> is formed as a single part allowing to be sandwiched between the first substrate <NUM> or rather the first hydrophobic surface <NUM> and the cover <NUM>. In doing so, in setting up the microfluidics system <NUM>, first the spacer <NUM> is placed onto the substrate <NUM> or rather the first hydrophobic surface <NUM>. Afterwards, the cover <NUM> is placed onto the spacer <NUM>.

The spacer <NUM> can be formed such to separate a plurality of working areas in the gap <NUM> provided between the first and second hydrophobic surfaces <NUM>, <NUM>. In this option, the spacer <NUM> can be formed as a planar component comprising recesses in areas which act as working areas in the gap <NUM> provided between the first and second hydrophobic surfaces <NUM>, <NUM>. Hence, the spacer <NUM> can act itself as a barrier used to delimit respective working areas. This barrier feature of the spacer <NUM> allows to prevent mixture of liquids and to prevent from cross-contaminations during handling. Additionally, the spacer <NUM> still acts to support the cover <NUM>. The cover <NUM> is placed on the first hydrophobic surface <NUM> with the gap <NUM> interposed. The gap <NUM> can be filled with liquid <NUM> introduced from the micro-container <NUM>. Otherwise, liquid <NUM> contained in said gap <NUM> can be withdrawn into the micro-container <NUM>.

In a second aspect of the second embodiment, the first hydrophobic surface <NUM> is comprised by a working film <NUM> that is reversibly placeable on the first substrate <NUM>. Further, the second hydrophobic surface <NUM> is comprised by the cover <NUM> that is configured as a rigid plate and to be accommodated on the working film <NUM>. In this aspect, the cover <NUM> comprises the spacer <NUM> for separating said first and second hydrophobic surfaces <NUM>,<NUM> when accommodating the cover <NUM> on the working film <NUM> which is placed on said first substrate <NUM> of the digital microfluidics system <NUM>. In a further option, the spacer <NUM> can be comprised by the working film <NUM>. Furthermore, the spacer <NUM> can be formed as a single component, acting itself as a barrier used to delimit at least two working areas onto the working film <NUM>.

According to a first aspect in the second embodiment as depicted in <FIG> on the left side, in a method of introducing liquid into the gap <NUM> of the digital microfluidics system <NUM>, said first hydrophobic surface <NUM> is irremovably comprised by said first substrate <NUM> and the second hydrophobic surface <NUM> is comprised by said cover <NUM> that is configured as a rigid plate. This method further comprises a sixth step of accommodating the cover <NUM> on the first substrate <NUM>, and a seventh step of separating said first and second hydrophobic surfaces <NUM>,<NUM> by a spacer <NUM> that is separately provided. In an alternative, the spacer <NUM> can be comprised by the cover <NUM>. In a further alternative, the spacer <NUM> can be comprised by the first substrate <NUM> of the digital microfluidics system <NUM>.

According to a second aspect in the second embodiment as depicted in <FIG> on the right side, in a method of introducing liquid into a gap <NUM> of a digital microfluidics system <NUM>, said first hydrophobic surface <NUM> being comprised by a working film <NUM> that is reversibly placeable on said first substrate <NUM> and the second hydrophobic surface <NUM> being comprised by said cover <NUM> that is configured as a rigid plate. The method comprises a sixth step of placing the working film <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM>. The method further comprises a seventh step of accommodating the cover <NUM> on the first substrate <NUM>; and an eighth step of separating said first and second hydrophobic surfaces <NUM>,<NUM> by a spacer <NUM> that is separately provided or that is comprised by the cover <NUM> or by the working film <NUM>.

In the first and second embodiments as depicted in <FIG>, a method of withdrawing liquid <NUM> from the gap <NUM> of the digital microfluidics system <NUM> for manipulating samples in liquid portions or droplets is provided, wherein the digital microfluidics system <NUM> comprises the first substrate <NUM> and the central control unit <NUM>, wherein said first substrate <NUM> comprises the array of electrodes <NUM>, and wherein said central control unit <NUM> is in operative connection to said electrodes for controlling the selection of individual electrodes <NUM> thereof and for providing a number of said electrodes with voltage for manipulating liquid portions or droplets by electrowetting. In said digital microfluidics system <NUM>, the working gap <NUM> with a gap height is located parallel to the array of electrodes <NUM> and in-between the first and second hydrophobic surfaces <NUM>,<NUM>, wherein the two hydrophobic surfaces <NUM>,<NUM> facing each other at least during operation of the digital microfluidics system <NUM>. The method comprises the steps of: (a) placing the cover <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM>, the cover <NUM> comprising on one side the second hydro-phobic surface <NUM> and on another side at least one micro-container interface <NUM>; said at least one micro-container interface <NUM> comprising at least one cone <NUM> with an inner surface and at least one fluidic access hole <NUM> formed into the cover <NUM> and interconnecting each cone <NUM> and the gap <NUM>; (b) providing an essentially uniform height of the gap <NUM> between said first and second hydrophobic surfaces <NUM>,<NUM>; (c) inserting the nozzle <NUM> of at least one micro-container <NUM> into at least one cone <NUM> of the micro-container interface <NUM> of the cover <NUM>; (d) creating a sealing form fit contact between the inner surface of the at least one cone <NUM> of the micro-container interface <NUM> and an outer surface of the nozzle <NUM> of the inserted at least one micro-container <NUM>; and (e) aspirating liquid from the gap <NUM> into the at least one micro-container <NUM> via the at least one fluidic access hole <NUM> formed into the cover <NUM>.

In the first embodiment, in a first aspect as depicted in <FIG> on the left side, a method of withdrawing liquid <NUM> from the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein the cover <NUM> is comprised by the disposable cartridge <NUM>, the disposable cartridge <NUM> comprises the working film <NUM> with the first hydrophobic surface <NUM> and the cover <NUM> comprises the second hydrophobic surface <NUM>, wherein the cover <NUM> of the disposable cartridge <NUM> being configured rigid or flexible, wherein at least one spacer <NUM> being attached to the cover <NUM>, the second hydrophobic surface <NUM> being separated or separable from said first hydrophobic surface <NUM> by said gap <NUM>, said working film <NUM> comprising a backside that is configured to touch an uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>. The method further comprises the steps of: (f) sealingly enclosing the gap <NUM> with the spacer <NUM>; (g) defining with the spacer <NUM> the height of the gap <NUM> between the first and second hydrophobic surfaces <NUM>,<NUM> of the disposable cartridge, and permanently separating the first and second hydrophobic surfaces <NUM>,<NUM>; and (h) positioning the disposable cartridge <NUM> on the array of electrodes <NUM> of the first substrate <NUM> of the digital microfluidics system <NUM>.

In the first embodiment, in a second aspect as depicted in <FIG> on the right side, a method of withdrawing liquid <NUM> from the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein the cover <NUM> is comprised by the disposable cartridge <NUM>, wherein the disposable cartridge <NUM> comprises the working film <NUM> with the first hydrophobic surface <NUM> and the cover <NUM> comprises the second hydrophobic surface <NUM>, wherein the cover <NUM> of the disposable cartridge <NUM> is configured rigid and the working film <NUM> of the disposable cartridge <NUM> is configured flexible; wherein at least one gasket <NUM> is attached to the cover <NUM> and outside of the gap <NUM> for separating said first and second hydrophobic surfaces <NUM>,<NUM>. The method further comprises the steps of: (f) positioning the disposable cartridge <NUM> on the array of electrodes <NUM> of the first substrate <NUM> of the digital microfluidics system <NUM>; (g) creating an underpressure between the backside of the working film <NUM> and the uppermost surface of the first substrate <NUM> of the digital microfluidics system <NUM>; and (h) spreading the working film <NUM> on the first substrate <NUM> of the digital microfluidics system <NUM> and establishing the gap height.

In the second embodiment as depicted in <FIG>, in a first aspect, a method of withdrawing liquid <NUM> from the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein said first hydrophobic surface <NUM> is irremovably comprised by said first substrate <NUM> and the second hydrophobic surface <NUM> is comprised by said cover <NUM> that is configured as a rigid plate. The method further comprises the steps of: (f) accommodating the cover <NUM> on the first substrate <NUM>; and (g) separating said first and second hydrophobic surfaces <NUM>,<NUM> by a spacer <NUM> that is separately provided or that is comprised by the cover <NUM> or by the first substrate <NUM> of the digital microfluidics system <NUM>.

In the second embodiment as depicted in <FIG>, in a second aspect, a method of withdrawing liquid <NUM> from the gap <NUM> of the digital microfluidics system <NUM> is provided, wherein said first hydrophobic surface <NUM> is comprised by a working film <NUM> that is reversibly placeable on said first substrate <NUM> and the second hydrophobic surface <NUM> is comprised by said cover <NUM> that is configured as a rigid plate. The method further comprises the steps of: (f) placing the working film <NUM> on the first substrate <NUM> of the digital micro-fluidics system <NUM>; (g) accommodating the cover <NUM> on the first substrate <NUM>; and (h) separating said first and second hydrophobic surfaces <NUM>,<NUM> by a spacer <NUM> that is separately provided or that is comprised by the cover <NUM> or by the working film <NUM>.

<FIG> depicts cross sectional views of micro-containers <NUM> different in size. The micro-container <NUM> is a disposable plastic micro-syringe comprising the tube <NUM> with the nozzle <NUM> formed integrally. The nozzle <NUM> comprises an aperture having a predefined diameter. The tube <NUM> sealingly receives the piston <NUM> which is guided inside the tube <NUM> in an axial direction for dispensing or aspirating liquid via the nozzle <NUM> of the micro-container <NUM>. The outer surface of the nozzle <NUM> is designed to create a sealing form fit contact with an inner surface of a cone comprised by the micro-container interface <NUM> (refer to <FIG>) of the cover <NUM>. This feature allows to minimize dead volume. The micro-container <NUM> can be made out of a cost-effective biocompatible material to be disposed of after a single use. In further examples, the micro-container <NUM> can be made out of polypropylene, polystyrene, polyethylene, polycarbonate, cyclic olefin copolymers (TOPAS), or cyclic olefin polymers (Zeonor). The micro-container <NUM> can be a high-capacity container having a capacity of 1000µl, 500µl, 100µl, for example. The micro-dimension of the aperture at the distal end of the nozzle <NUM> is ≤ <NUM> or ≤ <NUM>, for example, in order to prevent leakage of solutions out of the micro-container <NUM> because surface tension effects can dominate over hydrostatic pressure. The micro-container <NUM> can be made opaque to protect reagents sensitive to light from possible degradation (e.g. fluorophores, HPP substrate). In an example, the micro-container <NUM> is pre-filled with a liquid selected from a group comprising reagents, oil, buffers and samples.

In order to be gripped by a manifold, preferably an outer surface of the tube <NUM> of the micro-container <NUM> is provided with a first gripping portion <NUM>. In order to be gripped by a robot or an actuator, the distal end of the piston <NUM> of the micro-container <NUM> is provided with a second gripping portion <NUM>. The first and second gripping portions <NUM>,<NUM> preferably comprise an outer rim projecting radially from the outer surfaces of the tube <NUM> and piston <NUM>, respectively. In operation, applying a force to the first and second gripping portions <NUM>,<NUM> in a direction such to move them to each other causes dispensing liquid from the micro-container <NUM>, and in a direction such to move them from each other causes aspirating liquid into the micro-container <NUM>.

Further advantages of the micro-container <NUM> are as follows. The high capacity of the micro-container <NUM> allows consecutive partial injections of reagents into a disposable cartridge (refer e.g. <FIG>). The micro-container <NUM> can withdraw liquid from the gap of the disposable cartridge such to act as waste storage or for the recovery of treated samples that require further analysis onto a different instrument. Further, the injection of liquids, e.g. reagents pre-loaded into the micro-containers <NUM>, is computer controlled so that individual or multiple simultaneous injections can be performed whenever required. Furthermore, the volumes of liquid injected into the electrowetting cartridge can vary between 1µl-200µl, more preferably 10µl-100µl. A bolus of air can be added at the tip of the nozzle <NUM> of the micro-container <NUM> to isolate the reagent or its chemical components from a filler fluid during operation. This bolus of air can be injected with the reagent into the disposable cartridge if necessary. The micro-container <NUM> can be part of a collection kit in order to collect a sample (e.g. blood from a finger prick). The micro-dimension of the micro-container <NUM> allows proper loading of a sample into the micro-container <NUM> via capillary action. The micro-container <NUM> can contain lyophilized reagents that can be re-solubilized by aspirating buffer solution brought to a fluidic access hole of a disposable cartridge. Advantageously, such pre-packaged reagents would not require special handling (e.g. temperature) during transportation and storage.

<FIG> is a perspective view of a first embodiment of a manifold <NUM> equipped with a plurality of micro-containers <NUM> (refer to <FIG>). For the sake of a better overview, a single micro-container <NUM> (the right one in the Figure) is shown removed from the manifold <NUM>. The manifold <NUM> comprises a plurality of elongated micro-container receptacles <NUM> aligned to each other in parallel. Each of the receptacles <NUM> comprises elongated recesses formed into the manifold <NUM>, continuously. Each of said receptacles <NUM> is adapted to receive a micro-container <NUM>. In the exemplary embodiment as depicted in <FIG>, the micro-containers <NUM> are inserted into or rather coupled to the receptacles <NUM> by moving the first gripping portion <NUM> of each micro-container <NUM>, which first gripping portion <NUM> comprises an outer rim projecting radially from the outer surfaces of the tube, into a respective groove <NUM> formed into each of the receptacles <NUM> of the manifold <NUM>. In doing so, each of the micro-containers <NUM> can be received in and coupled to the manifold <NUM> in a releasable manner, at least in an axial direction of the micro-container <NUM>.

In the exemplary embodiment shown in <FIG>, at least one rim part of the first gripping portion <NUM> of the micro-container <NUM> is formed planar or rather flattened. This flattened portion is formed such to align with respective planar portions of the manifold <NUM> located in a region adjacent to the loaded micro-container <NUM>. In other words, the flattened portion of the first gripping portion <NUM> of the micro-container <NUM>, once inserted into the receptacle <NUM>, aligns with wall portions of the manifold <NUM>. The manifold <NUM> is adapted to receive a clip <NUM> attachable to the manifold <NUM> such to engage the planar rim part of the first gripping portion <NUM> of respective micro-containers <NUM> received in the receptacles <NUM> (refer e.g. to <FIG>). In doing so, the aligned portion of the manifold <NUM> can be engaged by means of the clip <NUM> attachable to the manifold <NUM>, as shown in <FIG>. The clip <NUM> is adapted to engage the planar rim part of the first gripping portion <NUM> of at least one micro-container <NUM> received in one of the receptacles <NUM>. In other words, the micro-containers <NUM> are secured to the manifold <NUM> by means of the clip <NUM>. In a first aspect, attachment of the clip <NUM> to the manifold <NUM> at least on one side of the clip <NUM> is a snap-fit connection.

In the examples shown e.g. in <FIG>, attachment of the clip <NUM> to the manifold <NUM> on both sides of the clip <NUM> is a snap-fit connection. In a non-shown further example, the clip <NUM> can be hinged to the manifold <NUM> on one lateral side thereof, wherein attachment of the clip <NUM> to the manifold <NUM> on the other side of the clip <NUM> is a snap-fit connection. In this example, the clip <NUM> is pivotally supported to the manifold <NUM> on one side. The other side or rather the non-hinged part of the clip <NUM> can be provided with a snap-fit means, e.g. a latch, adapted to engage a periphery portion of the manifold <NUM>. This feature preferably allows biased engagement of the micro-containers <NUM> in the manifold <NUM> by proper pushing or rather urging the micro-containers <NUM> into the grooves <NUM> of the manifold <NUM> (refer to <FIG>). In this example, said sleeve can abut against or rather be seated on ledges (refer to ledges <NUM> in <FIG>) formed on the front side of the manifold <NUM>. A detailed description of the ledges will be given below. In order to allow the micro-containers <NUM> to be released from the manifold <NUM>, the sleeve can be removed from the manifold <NUM> by pulling the sleeve from the manifold <NUM> in an upward direction.

Returning back to <FIG>, it is preferred that the manifold <NUM> further comprises a releasing lever <NUM> movably attached to the periphery of the manifold <NUM> such to be moved in an upwards and downwards direction. Said releasing lever <NUM> is for releasing latches (refer to <FIG>) snapped into a recess <NUM> formed into lateral wall portions of the manifold <NUM>. Said recess <NUM> can be formed elongated, recessed into the manifold <NUM> from the rear of the manifold <NUM>, for example. A region of the manifold <NUM> beneath the recess <NUM> can be provided with a protrusion <NUM>. The protrusion <NUM> can be formed such to not fully overlap the elongated recess <NUM>. A more detailed description of the recess <NUM> and the protrusion <NUM> will be provided in the following.

The releasing lever <NUM> can be snapped onto the periphery of the manifold <NUM> from the rear such to be clamped between endmost lateral sides of the front side and the whole back side of the manifold <NUM>. In other words, the releasing lever <NUM> is mounted to the manifold <NUM> such to still maintain the front side of the manifold <NUM> fully exposed to the outside. This feature still allows the micro-containers <NUM> to be inserted into and removed from the receptacles <NUM>. Further, the releasing lever <NUM> is clamped onto the manifold <NUM> in a region between the protrusions <NUM> and ledges <NUM> formed on partition walls between respective adjacent receptacles <NUM> on the front side of the manifold <NUM>. A more detailed description of the ledges <NUM> will be provided in the following. The releasing lever <NUM> is movable up and down in relation to the periphery of the manifold <NUM> in a range delimited by the protrusions <NUM> and the ledges <NUM>. Therefore, unintentional drop-off of the releasing lever <NUM> can be prevented.

As mentioned above, the manifold <NUM> allows reception of a plurality of micro-containers <NUM> (e.g., in the embodiment shown in <FIG> and <FIG>, the manifold <NUM> receives a total of six micro-containers <NUM>). Therefore, individually loading a disposable cartridge (refer to <FIG>) with single micro-containers <NUM> can be avoided. Hence, advantageously, the number of operations required from the user during instrument initialization is reduced. Further, while not shown, the manifold <NUM> loaded with the micro-containers <NUM> can comprise a registration feature to prevent improper installation onto the disposable cartridge, for example. Furthermore, while not shown, the manifold <NUM> can contain a sonication device to create homogeneous solutions prior to injection into the electrowetting cartridge. Such solutions can consist of suspensions of particles, and more specifically, magnetic beads. Sonication can also be used to disrupt cell membrane.

<FIG> is a perspective view of the manifold <NUM> as shown in <FIG> and <FIG> as well as a linear array of caps <NUM> for attachment thereof to the bottom of the manifold <NUM>. This attachment can be a releasable attachment. The linear array of caps <NUM> comprises a support <NUM> and a plurality of caps <NUM> mounted on the support <NUM>, the number of the caps <NUM> equals the number of micro-containers <NUM> insertable into the manifold <NUM>. In other words, the manifold <NUM> is adapted to receive at least one cap <NUM> attachable to the manifold <NUM> at a bottom side thereof, the at least one cap <NUM> being formed such to sealingly engage a nozzle of a micro-container <NUM> received in the manifold <NUM>.

Each of the caps <NUM> of the linear array of caps <NUM> is formed such to sealingly engage a nozzle of a micro-container <NUM> respectively received in the manifold <NUM>. In doing so, if the caps <NUM> are attached to the manifold <NUM>, a cone <NUM> comprised by each of the caps <NUM> receives a respective nozzle of a micro-container <NUM>. The inner surface of each cone <NUM> is formed such to provide a sealing form fit contact with an outer surface of the nozzle of a respective micro-container <NUM> inserted in the manifold <NUM>. In other words, the cones <NUM> reliably plug the micro-containers <NUM> against leakage of liquids. The caps <NUM> can be added to the respective nozzles of individual micro-containers <NUM> after reagents loading thereof. Therefore, accidental cross-contaminations or leakage of reagent into the environment during transportation or storage can be avoided.

As mentioned above, the attachment of the linear array of caps <NUM> to the manifold <NUM> at the bottom side thereof preferably is a releasable attachment, in particular a snap-fit connection. In doing so, the support <NUM> comprises latches <NUM> for releasable attachment of the support <NUM> to the manifold <NUM> and for temporary sealing form fit contact with an outer surface of nozzles of the micro-containers <NUM> received in the manifold <NUM> if the caps <NUM> are attached to the manifold <NUM>. The latches <NUM> are provided at outermost lateral sides of the support <NUM>. Said latches <NUM> each protrude upwards from an upper surface of the support <NUM>. Each of the latches <NUM> is adapted to snap into the recesses <NUM> formed into the lateral sides of the manifold <NUM> (refer to the above). If snapped-in, having regard to the example shown in <FIG>, rear side portions of the latches <NUM> abut against the protrusions <NUM>, respectively, formed into the lateral sides of the manifold <NUM>, as well (refer to the above). Further, front side portions of distal ends of the ledges <NUM> snapped into the recesses <NUM>, respectively, abut against respective faces of the recesses <NUM>. This feature reliably prohibits lateral movement as well as pivotal movement of the linear array of caps <NUM> and the manifold <NUM> in relation to each other, if the linear array of caps <NUM> is attached to the manifold <NUM>. Hence, a reliable and very firm connection is provided.

<FIG> is a perspective view of an exemplary trough <NUM> adapted to receive a manifold <NUM> by inserting it from the above, and <FIG> is a perspective view of the first embodiment of the trough <NUM> as shown in <FIG> equipped with the manifold <NUM> as shown e.g. in <FIG>. The trough <NUM> is capable of keeping the reagent inside the micro-containers <NUM> inserted into the manifold <NUM> at a specific temperature either passively (e.g. ice pack) or actively (e.g. circulating coolant, thermoelectric coolers). Once inserted into the trough <NUM>, the bottom portions of the ledges <NUM> formed on the partition wall portions of the manifold <NUM> on the front side thereof abut against the upper rim of the trough <NUM>. As can be seen in the <FIG>, the trough <NUM>, on a right lateral side thereof, is provided with a feeding connection <NUM> allowing applying tempering liquid into the interior of the trough <NUM> and an outlet connection <NUM> allowing withdrawing of the tempering liquid from the interior of the trough <NUM> into which a part of the micro-containers <NUM> accommodated in the manifold <NUM> are reaching. Hence, reliable cooling of liquid inserted into the micro-containers <NUM> is achieved.

Preferably, an assembly comprising the manifold <NUM> equipped with the micro-containers <NUM>, the nozzles of which are sealed by means of the linear array of caps <NUM>, is received into the trough <NUM>. This arrangement allows to keep liquids inside the micro-containers <NUM> at a specific temperature as well as to prevent leakage of the liquids out of the micro-containers or rather mixture of liquids leaked from different micro-containers <NUM>.

<FIG> is a perspective view of a first aspect of the disposable cartridge <NUM> (refer to <FIG>) equipped with a manifold <NUM> in which a plurality of micro-containers <NUM> are received, and <FIG> is a cross sectional view of the first aspect as shown in <FIG>. In the shown aspect, each of the micro-containers <NUM> is connected to a respective one of a plurality of mechanical actuators <NUM>. In particular, each mechanical actuator <NUM> is connected to a respective micro-container <NUM> via its second gripping portion <NUM> (e.g. refer to <FIG>). For example, the mechanical actuators <NUM> form part of or interface with a robotic arm (not shown). The arrangement shown in <FIG> allows for on-demand injection of liquids into e.g. the disposable cartridge <NUM>. In this arrangement, the injection of liquid into the disposable cartridge <NUM> is performed by positive displacement when the piston <NUM> of the micro-container <NUM> is pushed down by mechanical actuation of a respective one of the mechanical actuators <NUM>. Otherwise, the aspiration of liquid from the disposable cartridge <NUM> into the micro-container <NUM> is performed by negative displacement when the piston <NUM> of the micro-container <NUM> is pulled up by mechanical actuation of a respective one of the mechanical actuators <NUM>. Therefore, advantageously, no user operation is required and automatic processing of microfluidic assays is enabled. The integration of the disposable cartridge <NUM> with the robotic liquid handler allows the delivery of samples and reagents into the gap of the disposable cartridge <NUM> whenever needed and with a large range of volumes possible (e.g. <NUM>-1000µl).

The manifold <NUM> can be mounted to the cover <NUM>, e.g. of a disposable cartridge <NUM> by means of a removable snap-fit connection. As best shown in <FIG>, the cover <NUM> of a disposable cartridge <NUM> preferably is equipped with latches <NUM>, which are adapted to engage recesses formed into the lateral wall of the manifold <NUM> (refer to latches <NUM> shown in <FIG> and recesses <NUM> shown in <FIG>). Once the latches <NUM> are snapped into the recesses, removal of the manifold <NUM> from the disposable cartridge <NUM> is blocked.

In order to disengage the snap-fit connection, the releasing lever <NUM> (also refer to <FIG>), which is movably mounted to the manifold <NUM>, can be pulled downwards, as schematically shown on the right side of the <FIG>. In doing so, a bottom edge of the releasing lever <NUM> engages a sloped portion formed on the upper end portion of each of the latches <NUM>. A further movement of the releasing lever <NUM> downwards results to the bottom edge of the releasing lever <NUM> further slides along the sloped upper end portion of each of the latches <NUM>, which sliding consecutively urges the latches <NUM> outwards or rather in a direction away from the manifold <NUM> or rather out of the respective recesses thereof. In turn, this outward urging moves the latches <NUM> out of engagement with the recess. Once the latches <NUM> are totally disengaged or rather released from the respective recesses, the manifold <NUM> is free to be removed from a cover <NUM>, e.g. from a disposable cartridge <NUM> by simply pulling the manifold <NUM> upwards while the disposable cartridge <NUM> remains in place. It is to be noted that reinstallation of the manifold <NUM> to the disposable cartridge <NUM> requires the releasing lever <NUM> to be moved upwards, previously, as schematically shown on the left side of the <FIG>.

Alternatively and departing from the embodiments shown in the Figures, the manifold <NUM> (or a number of manifolds <NUM>) can be irremovably attached to or can be integrated into a cover <NUM> of all herein disclosed varieties, e.g. a cover <NUM> of a disposable cartridge <NUM> (not shown).

Claim 1:
A method of introducing liquid (<NUM>) into a gap (<NUM>) of a digital microfluidics system (<NUM>) for manipulating samples in liquid portions or droplets; the digital microfluidics system (<NUM>) comprising a first substrate (<NUM>) and a central control unit (<NUM>), wherein said first substrate (<NUM>) comprises an array of electrodes (<NUM>), and wherein said central control unit (<NUM>) is in operative connection to said electrodes for controlling the selection of individual electrodes (<NUM>) thereof and for providing a number of said electrodes with voltage for manipulating liquid portions or droplets by electrowetting; in said digital microfluidics system (<NUM>), a working gap (<NUM>) with a gap height is located parallel to the array of electrodes (<NUM>) and in-between first and second hydrophobic surfaces (<NUM>,<NUM>); the two hydrophobic surfaces (<NUM>,<NUM>) facing each other at least during operation of the digital microfluidics system (<NUM>),
characterized in that the method comprises the steps of:
(a) placing a cover (<NUM>) on the first substrate (<NUM>) of the digital microfluidics system (<NUM>), the cover (<NUM>) comprising on one side the second hydrophobic surface (<NUM>) and on another side at least one micro-container interface (<NUM>); said at least one micro-container interface (<NUM>) comprising a cone (<NUM>) with an inner surface and at least one fluidic access hole (<NUM>) formed into the cover (<NUM>) and interconnecting the cone (<NUM>) and the gap (<NUM>);
(b) providing an essentially uniform height of the gap (<NUM>) between said first and second hydrophobic surfaces (<NUM>,<NUM>);
(c) providing at least one micro-container (<NUM>) filled with prepackaged liquid reagents;
(d) inserting a nozzle (<NUM>) of the at least one micro-container (<NUM>) into the cone (<NUM>) of the micro-container interface (<NUM>) of the cover (<NUM>);
(e) creating a sealing form fit contact between the inner surface of the cone (<NUM>) of the micro-container interface (<NUM>) and an outer surface of the nozzle (<NUM>) of the inserted at least one micro-container (<NUM>); and
(f) dispensing liquid (<NUM>) by positive displacement from the at least one micro-container (<NUM>) into the gap (<NUM>) via the at least one fluidic access hole (<NUM>) formed in the cover (<NUM>), wherein the injection of liquid into the gap occurs by positive displacement when a piston of the micro-container is pushed down by a mechanical actuation.