EP2943279B1 - Système de manipulation d'échantillons sous forme de gouttelettes liquides - Google Patents

Système de manipulation d'échantillons sous forme de gouttelettes liquides Download PDF

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Publication number
EP2943279B1
EP2943279B1 EP13700642.5A EP13700642A EP2943279B1 EP 2943279 B1 EP2943279 B1 EP 2943279B1 EP 13700642 A EP13700642 A EP 13700642A EP 2943279 B1 EP2943279 B1 EP 2943279B1
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EP
European Patent Office
Prior art keywords
electrode
liquid droplet
electrodes
optical
droplet manipulation
Prior art date
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Application number
EP13700642.5A
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German (de)
English (en)
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EP2943279A1 (fr
Inventor
Travis Lee
Michael Benjamin FRANKLIN
Kailiang Wang
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Tecan Trading AG
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Tecan Trading AG
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Publication of EP2943279A1 publication Critical patent/EP2943279A1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427—Electrowetting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers

Definitions

  • the present invention relates to a liquid droplet manipulation system and to a cartridge with a polymer film for manipulating samples in liquid droplets thereon.
  • the liquid droplet manipulation system comprises such a cartridge, an electrode array supported by a substrate, and a central control unit for controlling the selection of individual electrodes and for providing them with individual voltage pulses for manipulating liquid droplets by electrowetting.
  • Material of interest is collected e.g. from a crime scene (in criminal forensics) or from a patient (for diagnostic purposes).
  • Such materials can be tissue samples (such as oral mucosa cells, hair follicles) or bodily fluids (such as blood, sputum, etc.).
  • This starting material then requires further processing to make nucleic acids or proteins available for the analysis.
  • a lysis step is initially applied for these purposes, involving for example the application of heat, a certain enzymatic activity, and/or the application of specific chemicals.
  • the cell lysis is followed by a purification of the nucleic acid or protein of interest from the additional cellular material.
  • nucleic acid amplification is typically achieved by the polymerase chain reaction (PCR). This method allows the amplification of specific, predefined nucleic acid sequences by the use of sequence-specific primer. Depending on the question to be solved, the amplified material might be further analyzed for example by sequencing.
  • PCR polymerase chain reaction
  • Automated liquid handling systems are generally well known in the art.
  • An example is the Freedom EVO ® robotic workstation from the present applicant (Tecan für AG, Seestrasse 103, CH-8708 Gurnnedorf, Switzerland).
  • This device enables automated liquid handling in a stand-alone instrument or in automated connection with an analytical system.
  • These automated systems typically require larger volumes of liquids (microliter to milliliter) to process. They are also larger systems that are not designed to be portable.
  • a portable device for lysing and/or purifying biological samples is known from WO 2007/061943 .
  • the processing of nucleic acids is performed within a cartridge chamber using electrodes arranged on the two sides, thus processing biological material by electrolysis, electroporation, electro-osmosis, electrical kinetic or resistive heating.
  • the cartridge further comprises sieving matrixes or membranes.
  • the number of probes that can be worked on is limited to four different wavelengths that an associated instrument can detect in parallel.
  • the cartridge itself can be placed into an integrated system comprising the required control elements and energy sources. Although this cartridge provides a system to at least partially control the sample processing electronically, intervention of an investigator or of technical lab staff is still required.
  • electrowetting refers to a method to move liquid droplets using arrays of microelectrodes, preferably covered by a hydrophobic layer.
  • a defined voltage 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.
  • 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 lest 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.
  • liquid processing can be carried out within considerably shorter time.
  • control of the liquid movement can be completely under electronic control resulting in automated processing of samples.
  • 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 US patent No. 5,486,337 . 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 and electrode array with an opposing surface with at least one ground electrode of is known from US 6,565,727 (a biplanar arrangement of electrodes).
  • Each surface of this device may comprise a plurality of electrodes.
  • the drive electrodes of the electrode array are preferably arranged in an interdigitated relationship with each other by projections located at the edges of each single electrode.
  • 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.
  • 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 liquid droplet manipulation instrument comprises at least one electrode array for inducing liquid droplet movements.
  • a substrate supporting the 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.
  • the system thus enables displacement of at least one liquid droplet from the at least one well through the 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.
  • EP 1722234 A2 discloses a device for transporting liquid and the use of ITO electrodes that are coated with a water-repellent film and WO 2011/084703 A2 shows a droplet actuator with hydrophobic coated substrate and an electrode path or array comprising a plurality of electrodes including an optically transparent detection electrode comprising a plated through hole configured for light transmission.
  • EP 2548646 A2 shows a liquid droplet manipulation system with an optical electrode adjacent to an individual electrode.
  • EP 2548646 A2 was published after the priority date of the present patent application and is therefore only available for novelty assessment pursuant Article 54(3) of the European Patent Convention.
  • the invention involves a system for liquid droplet manipulation, the liquid droplet manipulation system comprising at least one cartridge with a polymer working film for manipulating samples in liquid droplets thereon and a substrate with at least one electrode array and a central control unit for controlling the selection of individual electrodes of the electrode array(s) and for providing the electrodes with individual voltage pulses for manipulating liquid droplets by electrowetting, wherein the liquid droplet manipulation system is configured to receive on top of the electrodes the polymer working film for manipulating the samples in liquid droplets with the electrode array(s) when the polymer working film is placed on said electrode array(s), and wherein at least one selected individual electrode of the electrode array(s) of the liquid droplet manipulation system is configured to be penetrated by light of an optical detection system for the optical inspection or analysis of samples in liquid droplets that are located on the polymer working film, wherein the at least one selected individual electrode is configured as an optically transparent electrode or as an optical via electrode that comprises a through hole, and the optically transparent electrode or the optical via electrode is located in place of an individual
  • the liquid droplet manipulation system comprises a substrate and an electrode array, on top of which a cartridge with a working film can be positioned for manipulating samples in liquid droplets with an electrode array of the liquid droplet manipulation system when the working film of the cartridge is placed thereon.
  • the cartridge is characterized in that it comprises:
  • the Figure 1 shows a vertical cross-section through a frame structured cartridge 1 according to a first embodiment with a central opening 14 closed by a bottom portion 16, with a number of wells 5 and a working film 10 contacted by a peripheral spacer 9 that is configured as a separate peripheral element 9".
  • the cartridge 1 is almost in contact with the electrode array 20 of a system 40 for liquid droplet manipulation.
  • This cartridge 1 comprises a working film 10 for manipulating samples in liquid droplets with an electrode array 20 when the working film 10 of the cartridge 1 is placed on said electrode array 20.
  • This cartridge 1 also comprises a body 2, which body 2 preferably comprises an essentially flat lower surface 4.
  • the body 2 is configured as a frame structure 2" with a central opening 14.
  • the body 2 comprises an upper surface 3, a lower surface 4, and a number of wells 5 configured to hold therein reagents 6 or samples 6'.
  • the material of the body 2 is of an inert plastic material that is impermeable to liquids and that does not take up or interfere with the liquids or samples contained in the wells 5.
  • Preferred materials for injection molding of the body 2 in the form of a frame structure 2" comprise cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene, polystyrene, polycarbonate, and glass.
  • Preferred production techniques other than injection molding comprise cutting and/or punching of e.g. polytetrafluorethylene or polytetrafluorethen (PTFE).
  • This cartridge 1 also comprises a flexibly deformable top structure 7 that is impermeable to liquids and that is configured to seal a top side of the wells 5.
  • the flexibly deformable top structure 7 is configured as a flexible foil that is sealingly attached to the upper surface 3 of the frame structure 2".
  • the flexible foil preferably is made of an elastomeric material, such as a rubber or a thermoplastic elastomer (TPE) membrane and preferably is sealingly attached to the upper surface 3 of the frame structure 2" by welding.
  • the flexibly deformable top structure 7 is configured as a flexible top portion of the body 2 that is integrated in the frame structure 2" (not shown).
  • the body material preferably is TPE.
  • This cartridge 1 also comprises a piercable bottom structure 8 that is impermeable to liquids and that is configured to seal a bottom side of the wells 5.
  • the piercable bottom structure 8 is configured as a piercable bottom portion of the body 2 that is integrated in frame structure 2".
  • the body material preferably is TPE.
  • the piercable bottom structure 8 is configured as a piercable foil that is sealingly attached to the lower surface 4 of the frame structure 2" (not shown).
  • the piercable foil preferably is made of an elastomeric material, such as a rubber or a thermoplastic elastomer (TPE) membrane.
  • This cartridge 1 also comprises a working film 10 that is located below the lower surface 4 of the body 2,2".
  • the working film 10 is impermeable to liquids and comprises a hydrophobic upper surface 11, on which the droplets are to be moved by electrowetting techniques.
  • the working film 10 is configured as a monolayer of a hydrophobic material:
  • the monolayer of hydrophobic material is also electrically insulating (so that the working film 10 electrically isolates each one of the individual electrodes 44 of the electrode array 20).
  • the cartridge 1 can directly be placed with its working film 10 on top of the electrode array 20 without any need of an additional dielectric layer.
  • Preferred materials for producing such a preferred dielectric/hydrophobic working film 10 are selected from the group comprising fluorinated ethylene propylene (FEP) such as perfluorethylenepropylene copolymer; perfluoralcoxy polymers and copolymers (PFA); cyclic olefin polymers and copolymers (COP); and polyethylene (PE).
  • FEP fluorinated ethylene propylene
  • PFA perfluoralcoxy polymers and copolymers
  • COP cyclic olefin polymers and copolymers
  • PE polyethylene
  • the cartridge 1 must be placed with its working film 10 on top of the electrode array 20 with an additional dielectric layer located between the electrode array 20 and the working film 10 (see e.g. Fig. 5 ).
  • an additional dielectric layer could be attached to the lower surface of the working film 10 or to the upper surface or surface level 48 of the individual electrodes 44 (as in Fig. 5 ).
  • an additional dielectric layer could be provided as a separate dielectric sheet that is to be positioned on the electrode array 20 before the cartridge 1 is placed thereon with its working film 10 (not shown).
  • a preferred material for producing such a working film 10 of a monolayer of hydrophobic non-dielectric material is for example polytetrafluorethylene or polytetrafluorethen (PTFE).
  • the working film 10 is configured as a monolayer of electrically non-conductive material of which the upper surface 11 is treated to be hydrophobic.
  • the cartridge 1 can directly be placed with its working film 10 on top of the electrode array 20 without any need of an additional dielectric layer.
  • Such treatment can be coating the monolayer of electrically non-conductive material with silanes (Marcia Almanza-Workman et al. 2002).
  • the working film 10 is configured as a laminate comprising a lower layer and a hydrophobic upper layer, the lower layer being electrically conductive or non-conductive: Similar as shown in Fig. 1 , the laminate of the working film 10 preferably comprises a dielectric lower layer and a hydrophobic upper layer, so that the working film 10 electrically isolates each one of the individual electrodes 44 of the electrode array 20. Alternatively, a third layer of hydrophobic material can be laminated to the lower side of the dielectric layer so that a sandwich is formed comprising a dielectric layer that is located between two hydrophobic layers. In any case, the cartridge 1 can directly placed with its working film 10 on top of the electrode array 20 without any need of an additional dielectric layer.
  • Preferred material combinations for producing such a preferred laminate working film 10 comprising at least one dielectric and at least one hydrophobic layer are e.g. selected from fluorinated ethylene propylene (FEP) such as perfluorethylenepropylene copolymer for the hydrophobic layer and polyimides (PI) like Kapton ® of DuPont for the dielectric layer.
  • FEP fluorinated ethylene propylene
  • PI polyimides
  • the cartridge 1 must be placed with its working film 10 on top of the electrode array 20 with an additional dielectric layer located between the electrode array 20 and the working film 10.
  • an additional dielectric layer could be attached to the lower surface of the working film 10 or to the upper surface or surface level 48 of the individual electrodes 44 (not shown).
  • an additional dielectric layer could be provided as a separate dielectric sheet that is to be positioned on the electrode array 20 before the cartridge 1 is placed thereon with its working film 10 (see Figs. 10 and 11 ).
  • This cartridge 1 also comprises a peripheral spacer 9 that is located below the lower surface 4 of the body 2,2',2" and that connects the working film 10 to the body 2,2',2".
  • This cartridge 1 also comprises a gap 12 between the lower surface 4 of the body 2,2',2" and the hydrophobic upper surface 11 of the working film 10.
  • This gap 12 is defined by the peripheral spacer 9.
  • the peripheral spacer 9 is configured as a peripheral rim 9' that surrounds an area of the gap 12 and that is integrally formed with the body 2 (see Fig. 2 ).
  • Fig. 2 Alternatively and as shown in Fig.
  • the peripheral spacer 9 is configured as a separate peripheral element 9" that surrounds the gap 12 and that is attached to the lower surface 4 of the body 2 that here is configured as a frame structure 2".
  • the working film 10 preferably is attached to the separate peripheral element 9" of the frame structure 2".
  • the cartridge 1 comprises intermediate spacers 15 that are located within the area of the gap 12 and that are attached to the lower surface 4 of the body 2 of the frame structure 2".
  • These intermediate spacers preferably have the same height as the separate peripheral element 9" and preferably define the same gap dimension.
  • This cartridge 1 also comprises a number of piercing elements 13 that are located below piercable bottom structures 8 and that are configured to pierce the piercable bottom structures 8 for releasing reagents or samples 6,6' from the wells 5 into the gap 12.
  • the piercing elements 13 are located within the area of the gap 12 and are integrally formed with the spacer 9 that is configured as a separate ring-like element 9" and that surrounds the gap 12.
  • the piercing elements 13 are located below a well 5 or an intake recess and are configured to pierce at least the piercable bottom structure 8 when actuated by an actuating element 41 of a system 40 for liquid droplet manipulation.
  • the actuating elements 41 preferably are guided in their movements by a guiding channel 45.
  • the central opening 14 of the frame structure 2" is configured as a depression in the upper surface 3 of the body 2 leaving a bottom portion 16 of the body 2 that is integrally formed with the frame structure 2" to form the substantially flat lower surface 4 of the body 2. Therefore, it is shown in Fig. 1 that the gap 12 extends between the lower surface 4 of the body 2 and the upper, hydrophobic surface 11 of the working film 10.
  • the substrate 42 comprises at least one optical fiber 21 for bringing light to a droplet 23 (here only indicated in dotted lines) in the gap 12 and/or for guiding light away from a droplet 23 in the gap 12.
  • a so called bottom reading optical system is indicated by the optical fiber 21.
  • excitation light originating from a light source (not shown) can be brought through an individual electrode 44 that is optically transparent (not shown) or that comprises a through hole (shown). The excitation light then penetrates the working film 10 that needs to be optically transparent and enters the droplet 23 with sample material in it.
  • the sample material comprises a fluorophore
  • this fluorophore will emit fluorescence that then is detected by the optical bottom reading system and a detector connected to the latter.
  • the bottom reading system in the embodiment shown in Fig. 1 is configured to send excitation light to the sample and to receive and detect fluorescence emitted by the sample.
  • the optical fiber 21 is integrated into the substrate 42 of the electrode array 20 of the system 40 for the manipulation of droplets. This substrate also comprises electrical lines that link the individual electrodes 44 with a central control unit 43 of the system 40.
  • FIG. 2 shows a vertical cross-section through a cartridge 1 with a body 2 that is configured as a plate-like structure 2' according to a first example.
  • This cartridge 1 comprises a number of wells 5 and a working film 10 that is contacted to the body 2 by an integrated peripheral rim 9'.
  • the cartridge 1 is almost in contact with the electrode array 20 of a system 40 for liquid droplet manipulation
  • This cartridge 1 also comprises a working film 10 for manipulating samples in liquid droplets with an electrode array 20 when the working film 10 of the cartridge 1 is placed on said electrode array 20.
  • This cartridge 1 also comprises a body 2, which body 2 preferably comprises an essentially flat lower surface 4.
  • the body 2 is configured as a plate-like structure 2'.
  • the body 2 comprises an upper surface 3, a lower surface 4, and a number of wells 5 configured to hold therein reagents 6 or samples 6'.
  • the material of the body 2 preferably is of an inert plastic material that is impermeable to liquids and that does not take up or interfere with the liquids or samples contained in the wells 5.
  • the same plastic materials for injection molding of the body 2 as for the frame structure 2" are also preferred for producing the plate-like structure 2' of this example.
  • This cartridge 1 also comprises a flexibly deformable top structure 7 that is impermeable to liquids and that is configured to seal a top side of the wells 5.
  • the flexibly deformable top structure 7 is configured as a flexible top portion of the body 2 that is integrated in the plate-like structure 2'.
  • the material for injection molding of the body 2 and its flexible top portion preferably is TPE.
  • the flexibly deformable top structure 7 is configured as a flexible foil that is sealingly attached to the upper surface 3 of the plate-like structure 2'.
  • the flexible foil preferably is made of an elastomeric material, such as a rubber or a thermoplastic elastomer (TPE) membrane and preferably is sealingly attached to the upper surface 3 of the plate-like structure 2' by welding.
  • TPE thermoplastic elastomer
  • This cartridge 1 also comprises a piercable bottom structure 8 that is impermeable to liquids and that is configured to seal a bottom side of the wells 5.
  • the piercable bottom structure 8 is configured as a piercable foil that is sealingly attached to the lower surface 4 of the plate-like structure 2'.
  • This piercable foil preferably is made of an elastomeric material, such as a rubber or a thermoplastic elastomer (TPE) membrane.
  • the piercable bottom structure 8 is configured as a piercable bottom portion of the body 2 that is integrated in the plate-like structure 2' (not shown).
  • the body material preferably is TPE.
  • This cartridge 1 also comprises a working film 10 that is located below the lower surface 4 of the body 2,2".
  • the working film 10 is impermeable to liquids and comprises a hydrophobic upper surface 11, on which the droplets are to be moved by electrowetting techniques. All embodiments of the working film 10 as well as the additional dielectric layer as described in connection with Fig. 1 are also preferred for the cartridge depicted in Fig. 2 .
  • This cartridge 1 also comprises a peripheral spacer 9 that is located below the lower surface 4 of the body 2,2',2" and that connects the working film 10 to the body 2,2',2".
  • This cartridge 1 also comprises a gap 12 between the lower surface 4 of the body 2,2',2" and the hydrophobic upper surface 11 of the working film 10.
  • This gap 12 is defined by the peripheral spacer 9.
  • the peripheral spacer 9 preferably is configured as a peripheral rim 9' that surrounds an area of the gap 12 and that is integrally formed with the body 2.
  • the peripheral spacer 9 is configured as a separate peripheral element 9" that surrounds the gap 12 and that is attached to the lower surface 4 of the body 2 that here is configured as a frame structure 2".
  • the working film 10 preferably is attached to the peripheral rim 9' of the plate-like structure 2'.
  • the cartridge 1 comprises intermediate spacers 15 that are located within the area of the gap 12 and that are integrally formed with the plate-like structure 2'.
  • These intermediate spacers 15 preferably have the same height as the peripheral rim 9' and preferably define the same gap dimension.
  • This cartridge 1 also comprises a number of piercing elements 13 that are located below piercable bottom structures 8 and that are configured to pierce the piercable bottom structures 8 for releasing reagents or samples 6,6' from the wells 5 into the gap 12.
  • the piercing elements 13 are located within the area of the gap 12 and close to the peripheral rim 9'.
  • the piercing elements 13 here are attached to the peripheral rim 9' and/or to the lower surface 4 of the body 2 of the plate-like structure 2'.
  • the piercing elements 13 are located below a well 5 or an intake recess and are configured to pierce at least the piercable bottom structure 8 when actuated by an actuating element 41 of a system 40 for liquid droplet manipulation.
  • the actuating elements 41 preferably are guided in their movements by a guiding channel 45.
  • the cartridge 1 comprises at least one optical fiber 21 for bringing light to a droplet 23 (here only indicated in dotted lines) in the gap 12 and/or for guiding light away from a droplet 23 in the gap 12.
  • a so called top reading optical system is indicated by the optical fiber 21.
  • excitation light originating from a light source (not shown) can be directly brought into the droplet 23 with sample material in it. If the sample material comprises a fluorophore, this fluorophore will emit fluorescence that then is detected by the optical top reading system and a detector connected to the latter. Accordingly, the top reading system in the example shown in Fig.
  • the substrate 42 is configured to send excitation light to the sample and to receive and detect fluorescence emitted by the sample.
  • the optical fiber 21 is integrated into the body 2 of the cartridge 1.
  • the substrate 42 also comprises electrical lines that link the individual electrodes 44 with a central control unit 43 of the system 40.
  • Figure 3 shows a vertical cross-section through a frame structured cartridge 1 according to a second embodiment with a central opening 14 across the entire height of the body 2.
  • the cartridge 1 comprises a number of wells 5 and a working film 10 contacted by a spacer 9 that is configured as a separate peripheral element 9".
  • the cartridge 1 is almost in contact with the electrode array 20 of a system 40 for liquid droplet manipulation.
  • This cartridge 1 comprises a working film 10 for manipulating samples in liquid droplets with an electrode array 20 when the working film 10 of the cartridge 1 is placed on said electrode array 20.
  • This cartridge 1 also comprises a body 2, which body 2 preferably comprises an essentially flat lower surface 4.
  • the body 2 is configured as a frame structure 2" with a central opening 14 that extends across the entire height of the body 2.
  • the body 2 comprises an upper surface 3, a lower surface 4, and a number of wells 5 configured to hold therein reagents 6 or samples 6'.
  • the lower surface 4 of the frame structure 2" of the body 2 is not completely flat:
  • the body 2 comprises an outer part 53 that is extended downwards.
  • this embodiment comprises a separate peripheral element 9" that is downwards bent according to the lower surface of the body 2.
  • the substrate 42 which is adapted to this special lower surface of the cartridge 1, comprises a surface 49 which is offset to a surface level 48 of the electrodes 44 such that at least a part of the lower surface 4 of the body 2,2',2" or of the spacer 9 of the cartridge 1 to which the working film 10 is attached is movable beyond the surface level 48 of the electrodes 44 for stretching the working film 10 on the electrodes 44.
  • the material of the body 2 is of an inert plastic material that is impermeable to liquids and that does not take up or interfere with the liquids or samples contained in the wells 5.
  • the same plastic materials for injection molding of the body 2 as for the frame structure 2" in Fig. 1 are also preferred for producing the frame structure 2" of this embodiment.
  • This cartridge 1 also comprises a flexibly deformable top structure 7 that is impermeable to liquids and that is configured to seal a top side of the wells 5.
  • the flexibly deformable top structure 7 is configured as a flexible foil that corresponds to the flexible foil in Fig. 1 .
  • This cartridge 1 also comprises a piercable bottom structure 8 that is impermeable to liquids and that is configured to seal a bottom side of the wells 5.
  • the piercable bottom structure 8 is configured as a piercable cover layer 19.
  • This cover layer 19 is configured as a piercable foil that is sealingly attached to the lower surface 4 of the frame structure 2" in a way that the cover layer 19 closes the gap 12 on a side opposite to the working film 10.
  • the lower surface of the cover layer 19 is essentially flush with the lower surface 4 of the frame structure 2".
  • the cover layer 19 is electrically conductive and is hydrophobic at least on a surface directed to the gap 12.
  • the cover layer may also be chosen such that the material of the cover layer 19 is from an electrically conductive and hydrophobic material, e.g. PTFE.
  • a cartridge 1 is preferred that comprises an electrical ground connection 54 which is connected to the cover layer 19 and which is attachable to a ground potential source of the system 40 for liquid droplet manipulation.
  • This cartridge 1 also comprises a working film 10 that is located below the lower surface 4 of the body 2,2".
  • the working film 10 is impermeable to liquids and comprises a hydrophobic upper surface 11, on which the droplets are to be moved by electrowetting techniques. All embodiments of the working film 10 as well as the additional dielectric layer as described in connection with Figs. 1 and 2 are also preferred for the cartridge depicted in Fig. 3 .
  • This cartridge 1 also comprises a peripheral spacer 9 that is located below the lower surface 4 of the body 2,2',2" and that connects the working film 10 to the cover layer 19 and to the body 2,2',2".
  • This cartridge 1 also comprises a gap 12 between the cover layer 19 and the hydrophobic upper surface 11 of the working film 10.
  • This gap 12 is defined by the peripheral spacer 9.
  • the peripheral spacer 9 is configured as a separate peripheral element 9" that surrounds an area of the gap 12 (compare with Fig. 1 ).
  • the working film 10 preferably is attached to the separate peripheral element 9" of the frame structure 2".
  • the cartridge 1 comprises intermediate spacers 15 that are located within the area of the gap 12 and that are attached to the lower surface of the cover layer 19 and/or to the hydrophobic upper surface 11 of the working film 10.
  • These intermediate spacers 15 preferably have the same height as the separate peripheral element 9" and preferably define the same gap dimension.
  • This cartridge 1 also comprises a number of piercing elements 13 that are located below wells 5 or below an intake recess and that are configured to pierce the cover layer 19 for releasing reagents or samples 6,6' from the wells 5 or the intake recess into the gap 12.
  • the piercing elements 13 are located similarly than shown in Fig. 1 .
  • the piercing elements 13 are actuated by an actuating element 41 of a system 40 for liquid droplet manipulation.
  • the actuating elements 41 preferably are guided in their movements by a guiding channel 45.
  • the central opening 14 of the frame structure 2" is configured as a through hole from the upper surface 3 to the lower surface 4 of the body 2 e 2".
  • the cover layer 19 forms the substantially flat lower surface 4 of the body 2.
  • the substrate 42 comprises at least one optical fiber 21 for bringing light to a droplet 23 (here only indicated in dotted lines) in the gap 12 and/or for guiding light away from a droplet 23 in the gap 12.
  • a window 22 in the cover layer 19 it may be preferred to provide a window 22 in the cover layer 19 at a place that is opposite the gap 12 and in register with the entrance/exit opening of the optical fiber 21.
  • bottom reading (compare with Fig. 1 ) and/or top reading (compare with Fig. 2 ) is enabled by the second embodiment of Fig. 3 .
  • the optical fiber 21 is integrated into the substrate 42 of the electrode array 20 of the system 40 for the manipulation of droplets.
  • This substrate also comprises electrical lines that electrically connect the individual electrodes 44 with a central control unit 43 of the system 40.
  • Figure 4 shows a vertical cross-section through the frame structured cartridge 1 according to the second embodiment of Fig. 3 .
  • the cartridge 1 is in contact with the electrode array 20 of a system 40 for liquid droplet manipulation.
  • the piercable bottom structure in the form of a cover layer 19 is opened for one well 5 and some of its content is pressed into the gap 12 between the working film 10 and the cover layer 19.
  • the substrate 42 here comprises an abutment surface 47 which is offset to a surface level 48 of the electrodes 44 such that a separate peripheral element 9" of the cartridge 1 to which the working film 10 is attached, is movable beyond the surface level 48 of the electrodes 44 for additionally stretching the working film 10 on the electrodes 44.
  • a clamping mechanism 52 presses the cartridge 1 and its working film 10 onto the surface 48 of the electrodes 44 and onto the surface 49 of the substrate 42.
  • Figure 5 shows a vertical cross-section through a frame structured cartridge 1 according to a second example with a central opening 14 across the body 2, with a number of wells 5 and a working film 10 contacted by a separate peripheral spacer element 9".
  • the cartridge 1 is in contact with the electrode array 20 of a system 40 for liquid droplet manipulation.
  • the piercable bottom structure 8 of one well (the intake recess 25) is opened and some of its content is pressed into the gap 12 between the working film 10 and a cover layer 19 that is configured as a rigid cover 17 here.
  • the material for this rigid cover preferably is Mylar ® , a transparent, flexible polyester foil on the basis of polyethylene tereph-thalat from DuPont.
  • the rigid cover 17 may be coated on its underside with a layer of indium tin oxide (ITO) in order to provide the rigid cover 17 with an electrically conductive layer that can be connected to a ground potential source of the system 40 for liquid droplet manipulation.
  • ITO indium tin oxide
  • This Fig. 5 also depicts a system 40 for liquid droplet manipulation that comprises a cartridge 1 and an electrode array 20.
  • This cartridge 1 comprises a working film 10 for manipulating samples in liquid droplets 23 with an electrode array 20 when the working film 10 of the cartridge 1 is placed on said electrode array 20.
  • This cartridge 1 also comprises a body 2, which body 2 preferably comprises an essentially flat lower surface 4, which is built by rigid cover 17 here.
  • the body 2 is configured as a frame structure 2" with a central opening 14 that extends across the entire height of the body 2.
  • the body 2 comprises an upper surface 3, a lower surface 4, and a number of wells 5 and intake recesses 25 configured to hold therein reagents 6 or samples 6'.
  • the material of the body 2 is of an inert plastic material that is impermeable to liquids and that does not take up or interfere with the liquids or samples contained in the wells 5.
  • the same plastic materials for injection molding of the body 2 as for the frame structure 2" in Figs. 1, 3, and 4 are also preferred for producing the frame structure 2" of this example.
  • This cartridge 1 also comprises a flexibly deformable top structure 7 that is impermeable to liquids and that is configured to seal a top side of the wells 5.
  • the flexibly deformable top structure 7 is configured as a flexible foil that corresponds to the flexible foil in the Figs. 1, 3, and 4 .
  • This cartridge 1 also comprises a piercable bottom structure 8 that is impermeable to liquids and that is configured to seal a bottom side of the wells 5 and intake recesses 25.
  • the piercable bottom structure 8 is configured as a piercable foil that is sealingly attached (e.g. by welding) to the lower surface 4 of the body 2.
  • This piercable foil preferably is made of an elastomeric material, such as a rubber or a thermoplastic elastomer (TPE) membrane.
  • the piercable bottom structure 8 is configured as a piercable bottom portion of the body 2 that is integrated in the plate-like structure 2' (compare Fig. 1 ).
  • the body material preferably is TPE.
  • the rigid cover 17 comprises cover holes 18, through which the piercing elements 13 easily reach the piercable foil.
  • the working film 10 is flexible so that no leaking out of liquids from the gap 12 has to be expected. All embodiments of the working film 10 as well as the additional dielectric layer as described in connection with the Figs. 1 to 4 are also preferred for the cartridge depicted in Fig. 5 .
  • the substrate 42 which is adapted to this flat lower surface of the cartridge 1, comprises a surface 49 which is flush with a surface level 48 of the electrodes 44 such that the working film 10 is stretched on the electrodes 44.
  • An electrically insulating film, layer or cover 50 is applied to the surface 48 of the electrodes 44 and to the surface 49 of the substrate 42.
  • This electrically insulating film, layer or cover 50 preferably is a dielectric layer that irremovably coats the electrodes 44 and substrate 42 of the system 40 for liquid droplet manipulation. It is however also preferred to provide an additional dielectric layer as a removable electrically insulating layer or cover 50 that can be replaced when needed.
  • the spacers 9,15 and piercing elements 13 of this cartridge 1 correspond with the spacers 9,15 and piercing elements 13 in Fig.1 and define a gap 12 between the rigid cover 17 and the hydrophobic upper surface 11 of the working film 10.
  • the piercing elements 13 are actuated by an actuating element 41 of a system 40 for liquid droplet manipulation.
  • the actuating elements 41 preferably are guided in their movements by a guiding channel 45.
  • the rigid cover 17 has essentially the same extension as the fame structure 2" and comprises a number of holes 18 located below the wells 5.
  • the holes 18 have a size and shape sufficient to allow bended piercing elements 13 to abut and pierce a respective piercable bottom structure 8 of a well 5.
  • the cartridge 1 comprises a rigid cover 17 and a cover layer 19 (the latter replacing the piercable foil as a piercable bottom structure 8).
  • the rigid cover 17 and the cover layer 19 are attached to the frame structure 2" in a way that the rigid cover 17 closes the gap 12 on a side opposite to the working film 10, a lower surface of the rigid cover 17 being essentially flush with the lower surface 4 of the frame structure 2".
  • the cover layer 19 (not shown in Fig. 5 ) preferably is placed between the rigid cover 17 and the lower surface 4 of the body 2.
  • the actuating elements 41 are configured as plungers that are slidingly movable in guiding channels 45 and that are agitated by an agitation mechanism 46. It also preferred that the agitation mechanism 46 for agitating the actuating elements 41 is configured as one of a wax pump bladder, a solenoid driven or clamping mechanism driven lever 51. It is further preferred that the agitation mechanism 46 for agitating the actuating elements 41 is configured as a clamping mechanism driven lever 51 and that the clamping mechanism 52 being hand driven and configured to press the body 2,2',2" of a cartridge 1 onto the substrate 42 and electrode array 20 of the system 40 for liquid droplet manipulation. Alternately, the clamping mechanism 52 is motor driven.
  • the Figure 6 shows a 3D top view of a frame-like cartridge 1 according to the second embodiment or second example with an intake device 26 in a passive position.
  • the body 2,2" of the cartridge 1 preferably comprises a specimen intake 24 that comprises an intake recess 25 and an intake device 26, the intake device 26 being at least partially positionable in an active position in the intake recess 25.
  • This specimen intake 24 is configured to introduce a buccal swab head 55 or other solid material comprising a sample to investigate.
  • the Fig. 6 also shows in the cross bar of the body 2 on the right side of the cartridge a number of wells 5 of different size for pre-depositing reagents and other liquids like wash fluids etc.
  • a very long well 5 which is configured to take up pre-deposited oil.
  • the oil can be used for filling the gap 12 prior to enter sample drops into the gap 12.
  • Complete filling of the gap 12 with an oil that is not miscible with the samples that normally are contained in a hydrous droplet and that is inert (e.g. silicon oil) is optional.
  • the size of the wells 5 can be chosen according to the actual need for carrying out particular assays.
  • a flexibly deformable top structure 7 that is configured as foil impermeable to liquids seals the top side of the wells 5.
  • the flexible foil is sealingly attached to the upper surface 3 of the frame structure 2" by laser welding for example.
  • an alternative intake recess 25' for introducing a sample of body fluid (like blood, saliva, etc.).
  • This alternative intake recess 25' preferably is sealed on its top side by a foil that is impermable to liquids, but that is also piercable with a needle of a medical syringe and that is flexible for being pushed by a piston-like actuating element for bringing the sample into the gap 12 of the cartridge 1 after the piercable bottom structure 8 has been pierced from the bottom side of the cartridge 1 with a piercing element 13.
  • the material for the foil that seals the top side of the alternative intake recess 25' preferably is rubber.
  • a frit 56 that is located in a channel which reaches down to the lower surface 4 of the body 2 and that preferably is combined with a semi-permeable membrane (not shown) is depicted.
  • This frit 56 and the channel serves as a vent for the gap 12 as soon as a piercable bottom structure 8 that sealingly closes the bottom of the channel has been pierced from the bottom side of the cartridge 1 with a piercing element 13.
  • intermediate spacers 15 can be seen through the optically transparent rigid cover 17 or cover layer 19. Although all intermediate spacers 15 drawn here are of equal size and round shape, and although these intermediate spacers 15 are distributed over the gap 12 at equal distances, the shape, size and distribution of these intermediate spacers 15 can be chosen as needed, if the intended electrowetting movements of the droplets 23 are not compromised.
  • the Figure 7 shows a bottom view of a frame-like cartridge 1 according to the second embodiment or second example of Fig. 6 with an intake device 26 in a passive position.
  • the working film 10 has been removed here so that the spacer 9 configured as a peripheral element 9" is visible.
  • the peripheral element 9" here is bordered by a downward extension 57 of the body 2.
  • This downward extension 57 of the body 2 in combination with the lower surface of the working film 10 (that is attached to the peripheral element 9" preferably provides the entire cartridge with a flat lower surface.
  • the downward extension 57 of the body 2 is flush with the peripheral element 9" and the working film 10 is attached to the working film 10 and as well to the downward extension 57 of the body 2.
  • piercing elements 13 can be seen here. Depending from the size of the well 5 above, the size and number of the piercing elements 13 can vary: i.e. for the oil containing well, three piercing elements 13 are depicted (see lower left); for the two largest wells that contain reagents, two piercing elements 13 are depicted (see upper right); and for the smaller wells containing reagents, only one piercing element 13 are depicted (see lower right).
  • the piercing element 13 that is configured to pierce the piercable bottom structure 8 below the intake recess 25 is shown on the left side of the top bar of the body 2.
  • the shown number, size and shape of these piercing elements 13 is only exemplary here and can vary according to actual needs.
  • the shape, size and distribution of the intermediate spacers 15 can be chosen as needed, if the intended electrowetting movements of the droplets 23 are not compromised.
  • the Figure 8 shows detailed 3D views of the specimen intake 24 of a frame-like cartridge 1 according to the second embodiment or second example.
  • Fig. 8A shows a semi cross-section of the specimen intake 24 of the frame-like cartridge with a partially inserted intake device 26 in the active position.
  • the intake device 26 preferably comprises a cylinder tube 27 with a first end 28 and with a second end 29, a plunger 30 that is insertable on the first tube end 28 and that is movable in the cylinder tube 27, and a sealing foil 31 that sealingly closes the second end 29 of the cylinder tube 27.
  • a pre-deposit of lysis buffer is provided in the space inside the cylinder tube 27 and between the plunger 30 and the sealing foil 31, a pre-deposit of lysis buffer is provided.
  • a frit 56 is also visible.
  • This frit 56 separates the part of the intake recess 25 (the outer chamber) in which the sample carrier, such as a buccal swab head 55, is placed for lysis of cellular material and the part of the intake recess 25 (the inner chamber) where the lysate is pressed into after the lysis.
  • the intake device 26 obviously has been moved from the passive position (see Figs. 6 and 7 ) to the active position, where the intake recess 25 of the cartridge 1 is located.
  • a flexibly deformable top structure 7 that is configured as a foil and that is impermeable to liquids seals the top side of intake recess 25.
  • the flexible foil is sealingly attached to the upper surface 3 of the frame structure 2" by laser welding for example.
  • Fig. 8B shows a semi cross-section of the specimen intake 24 of the frame-like cartridge 1 and of the partially inserted intake device 26 in the active position.
  • the situation depicted here is the following:
  • the Figure 9 shows a top view of an electrode layout or printed circuit board (PCB) of a system 40 for liquid droplet manipulation.
  • This particular electrode array 20 of the system 40 is configured for receiving a frame-like cartridge 1 according to the second embodiment or second example. Accordingly, the shape of the cartridge 1 with its central opening 14 is indicated in longer dashed lines here. The shape of the wells 5 and intake recess 25 is indicated in shorter dashed lines.
  • This electrode array 20 is particularly configured to match for the lysis of cellular material, for the extraction and PCR amplification of DNA fragments, for the hybridization experiments for genotyping, and for the optical detection. Four alignment marks in the corners of the electrode array facilitate alignment of the array.
  • the entire gap 12 is flooded with silicon (Si) oil. Then (see top right), from the intake recess 25 lysate (with or without beads) is entering the gap 12.
  • a first large electrode that is accompanied by a second large electrode.
  • the second large electrode in each case has a cut out, where the first of a row of individual electrodes 44 is placed.
  • a droplet of lysate and of pure wash liquid are moved by electrowetting to the wash zone where these droplets are mixed and washed and the magnetic beads and attached non-important sample parts are moved to a first waste zone, which is provided by a very large electrode.
  • master mix portions A and/or B can be added to the sample droplet.
  • a droplet is moved to the zone for polymerase chain reaction (PCR) where the nucleic acids contained in the sample droplet are amplified according to techniques known per se.
  • the PCR zone comprises at least two heater zones with a different temperature (e.g. 35 °C and 95 °C) for annealing and separating the strands of the nucleic acids.
  • a single ample drop with amplified nucleic acids is split into two smaller droplets at a splitting zone that preferably is characterized by the particular shape and arrangement of electrodes as depicted.
  • a splitting zone that preferably is characterized by the particular shape and arrangement of electrodes as depicted.
  • both of these two sample droplets are individually diluted with hybridization buffer and up to eight identical droplets are produced from each one of these two split sample droplets.
  • the twice eight sample droplets are subjected to hybridization according to techniques known per se. Following hybridization, the added, non-hybridized material is thoroughly washed away and discarded in a nearby second waste zone (which again is provided by a very large electrode).
  • Each one of the sixteen sample droplets is then individually moved (with electrowetting again) to a detection zone, where (using bottom reading, top reading, or a mixture or combination of both) the hybridized samples are optically analyzed.
  • the samples are discarded to the first waste zone and the "electrowetting path" provided by a large row of individual electrodes 44 is washed and cleaned a sodium hydroxide solution (NaOH) and optionally with a special wash solution.
  • NaOH sodium hydroxide solution
  • the cartridge 1 (together with the samples and the waste in it) is safely discarded so that nobody of the laboratory personnel is endangered by its contents. Then, the next cartridge 1 is pressed onto the electrode array 20 and the next experiments can be performed.
  • a large number of contact points are seen. Individual electric lines contact each electrode with one of these contact points.
  • heaters located in the substrate 42 of the system 40 are also connected to some of these contact points. All contact points are connected with the central control unit 43 which controls all necessary activations of e.g. heaters, plungers 41 etc. and of all electrical potentials of the electrodes that are required.
  • On each side of the electrode array is also provided a separate contact point for contacting with ground potential source of the central control unit 43.
  • the system 40 for liquid droplet manipulation comprises a substrate 42 with an electrode array 20 and a central control unit 43 for controlling the selection of individual electrodes 44 of the electrode array 43 and for providing the electrodes 44 with individual voltage pulses for manipulating liquid droplets 23 by electrowetting.
  • the system 40 is configured to receive on top of the electrodes 44 the working film 10 of a cartridge 1 according to the present invention.
  • the system 40 can be a stand alone and immobile unit, on which a number of operators is working with cartridges 1 that they bring along.
  • the system 40 thus may comprise a number of substrates 42 and a number of electrode arrays 20, so that a number of cartridges 1 can be worked on simultaneously and/or parallel.
  • the number of substrates 42, electrode arrays 20, and cartridges 1 may be 1 or any number between e.g. 1 and 100 or even more; this number e.g. being limited by the working capacity of the central control unit 43.
  • the system 40 can be implemented as a hand held which only comprises and is able to work with a single cartridge 1. 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.
  • the Figure 10 shows a partial top view of two electrode arrays 20, each one being equipped with two selected individual electrodes 44 that are configured as optical via electrodes 61 for the optical inspection or analysis of samples in liquid droplets 23.
  • Fig. 10A shows two optical via electrodes 61 of a first embodiment with activated or deactivated flanking electrodes 63 in a triangular shape.
  • Fig. 10B shows two optical via electrodes 61 of a second embodiment with activated or deactivated flanking electrodes 63 in a rectangular shape.
  • This liquid droplet manipulation system 40 comprises a substrate 42 with two electrode arrays 20 and with a central control unit 43 that is configured for controlling the selection of individual electrodes 44 of the electrode arrays 20 and for providing these electrodes 44 with individual voltage pulses for manipulating liquid droplets 23 by electrowetting.
  • the liquid droplet manipulation system 40 is configured to receive on top of the electrodes 44 a working film 10 (here, two individual working films 10 are shown) for manipulating samples in liquid droplets 23 with the electrode arrays 20 when the working film 10 is placed on at least one of said electrode arrays 20.
  • a working film 10 can have a smaller size that only covers a part of an electrode array 20 or a larger size that covers more than one electrode arrays (depending on the actual assay to be carried out).
  • at least one selected individual electrode 44 of the electrode arrays 20 of the liquid droplet manipulation system 40 is configured to be penetrated by light of an optical detection system for the optical inspection or analysis of samples in liquid droplets 23 that are located on the working film 10.
  • the two selected individual electrodes 44 are configured as optical via electrodes 61, each of which comprising a through hole 62.
  • An electrically insulating film or cover 50 is placed over the electrode arrays 20 in order to provide electrical insulation between the individual electrodes 44 of the electrode path 65 and between the liquid droplets 23 and the electrodes 44.
  • the working film is removable (peelable) from the insulating film or cover 50 and will be replaced each time a new experiment or assay is to be carried out.
  • the electrode path 65 shown is a closed loop; the electrode path 65 can be much more complex (e.g. comprising braches, crossings, arrays, reservoirs, and waste sinks) as exemplary can be seen in Fig. 9 .
  • the optical via electrodes 61 are accompanied by flanking electrodes 63 in the form of two similar orientated triangles, situated laterally to the optical via electrodes 61 and also connected to the electrode selection unit 64 of the central control unit 43 of the liquid droplet manipulation system 40 like all individual electrodes 44 and like the optical via electrodes 61.
  • flanking electrodes 63 in the form of two similar orientated triangles, situated laterally to the optical via electrodes 61 and also connected to the electrode selection unit 64 of the central control unit 43 of the liquid droplet manipulation system 40 like all individual electrodes 44 and like the optical via electrodes 61.
  • Fig. 10A shows two optical via electrodes 61 of a first embodiment with activated or deactivated flanking electrodes 63. The activated electrodes are shown in grey, the inactivated electrodes or electrodes kept at ground potential are shown in white.
  • This liquid droplet manipulation system 40 comprises a substrate 42 with two electrode arrays 20 and with a central control unit 43 that is configured for controlling the selection of individual electrodes 44 of the electrode arrays 20 and for providing these electrodes 44 with individual voltage pulses for manipulating liquid droplets 23 by electrowetting.
  • both flanking electrodes 63 of a single optical via electrode 61 are electrically connected with each other so that they always exhibit the same electrical potential (see right side).
  • a liquid droplet 23 exhibits an elliptic shape if the flanking electrodes 63 are activated (see on the left), and that a liquid droplet 23 exhibits an circular shape if the flanking electrodes 63 are deactivated (see on the right). Drifting off of the droplet 23 from the site of the through hole 62 of the optical via electrode 61 was not observed when the flanking electrodes 63 were deactivated.
  • the optical via electrodes 61 are accompanied by flanking electrodes 63 in the form of four similar orientated rectangles, pairwise situated laterally to the optical via electrodes 61 and also connected to the electrode selection unit 64 of the central control unit 43 of the liquid droplet manipulation system 40 like all individual electrodes 44 and like the optical via electrodes 61.
  • flanking electrodes 63 in the form of four similar orientated rectangles, pairwise situated laterally to the optical via electrodes 61 and also connected to the electrode selection unit 64 of the central control unit 43 of the liquid droplet manipulation system 40 like all individual electrodes 44 and like the optical via electrodes 61.
  • the activated electrodes are shown in grey, the inactivated electrodes or electrodes kept at ground potential are shown in white.
  • the Figure 11 shows a partial top view of two electrode arrays 20, each one being equipped with two selected individual electrodes 44 that are configured for the optical inspection or analysis of samples in liquid droplets 23.
  • Fig. 11A shows two optical via electrodes 61 of a third and fourth embodiment without flanking electrodes.
  • Fig. 11B shows two optically transparent electrodes 60, one located within a grid-like electrode array 26 and one located within a single electrode path 65.
  • This liquid droplet manipulation system 40 comprises a substrate 42 with two electrode arrays 20 and with a central control unit 43 that is configured for controlling the selection of individual electrodes 44 of the electrode arrays 20 and for providing these electrodes 44 with individual voltage pulses for manipulating liquid droplets 23 by electrowetting.
  • the liquid droplet manipulation system 40 is configured to receive on top of the electrodes 44 a working film 10 (here, two individual working films 10 are shown) for manipulating samples in liquid droplets 23 with the electrode arrays 20 when the working film 10 is placed on at least one of said electrode arrays 20.
  • a working film 10 can have a smaller size that only covers a part of an electrode array 20 or a larger size that covers more than one electrode arrays (depending on the actual assay to be carried out).
  • at least one selected individual electrode 44 of the electrode arrays 20 of the liquid droplet manipulation system 40 is configured to be penetrated by light of an optical detection system for the optical inspection or analysis of samples in liquid droplets 23 that are located on the working film 10.
  • the two selected individual electrodes 44 are configured as optical via electrodes 61, each of which comprising a through hole 62, and each of which having no flanking electrodes 63 nearby.
  • the two selected individual electrodes 44 are configured as optically transparent electrodes 60, each of which having no flanking electrodes 63 nearby.
  • Electrode path 65 is a closed loop and the grid-like electrode array 66 is rather small.
  • the electrode path 65 can be much more complex (e.g. comprising braches, crossings, arrays, reservoirs, and waste sinks) and the grid-like electrode array 66 much larger. Combinations of electrode paths 65 and grid-like electrode arrays 66 are also possible, as exemplary can be seen in Fig. 9 .
  • All individual electrodes 44 are operatively connected with the electrode selection unit 64 of the central control unit 43 of the liquid droplet manipulation system 40.
  • the electrode selection unit 64 For simplicity of the drawing, only a few of the electrical connections between the electrode selection unit 64 and the individual electrodes 44 present on (or in) the substrate 42 are drawn.
  • the activated electrodes are shown in grey, the inactivated electrodes or electrodes kept at ground potential are shown in white.
  • the optical via electrode 61 of the third embodiment is divided into two partial electrodes, each of which comprising a part of the through hole 62.
  • These two partial electrodes can be individually connected to the electrode selection unit 64 so that they can be individually activated or deactivated; this has the advantage of shifting a liquid droplet 23 more towards the adjacent electrode 44 of the electrode path 65, when only one of these partial electrodes is activated. In consequence, transportation of the liquid droplet 23 is facilitated.
  • these two partial electrodes of the optical via electrode 61 are electrically connected to each other so that only one connection exists with the electrode selection unit 64, consequently both so partial electrodes are simultaneously activated or deactivated.
  • the optical via electrode 61 of the fourth embodiment comprises a though hole 62 and lateral wings that at last partially surround an adjacent electrode of the electrode path 65. Activating this optical via electrode 61 of the fourth embodiment (including its wings) causes shifting of a liquid droplet 23 more towards the adjacent electrode 44 of the electrode path 65 (in the upward direction in Fig. 11A ), which facilitates transportation of the liquid droplet 23. In the opposite transportation direction, activating this optical via electrode 61 of the fourth embodiment (including its wings) facilitates moving of a liquid droplet 23 onto the optical via electrode 61 and its though hole 62.
  • a liquid droplet 23 exhibits a slightly elliptic shape.
  • a single optically transparent electrode 60 situated within a grid-like electrode array 66 is activated; thus, a single droplet 23 can be analyzed with the optics of the liquid droplet manipulation system 40.
  • an array of optically transparent electrodes 60 could be located within a grid-like electrode array 66 and all simultaneously activated; thus, a larger aggregate of single droplets 23 can be analyzed.
  • a single optically transparent electrode 60 situated within an electrode path 65 is activated; thus, a single droplet 23 can be analyzed with the optics of the liquid droplet manipulation system 40.
  • the diameter of the through hole 62 strongly depends on the actual size and shape of the respective optical via electrode 61 and is preferably as large as possible with the provision that the optical via electrode 61 still affects a liquid droplet 23 as required when being provided with individual voltage pulses for manipulating liquid droplets 23 by electrowetting. It is reasonable to relate the respective areas of an optical via electrode 61 and its through hole 62 to each other.
  • An optical via electrode 61 with square shape of 1.5 x 1.5 mm has an area of 2.25 mm 2 and a circular through hole 62 of 1 mm diameter has an area of 0.79 mm 2 ; of 1,1 mm diameter has an area of 0.95 mm 2 , and of 1.25 mm diameter has an area of 1.23 mm 2 .
  • the area ratio (optical via electrode 61: through hole 62) preferably is from 1.8 to 2.8; most preferably is 2.4.
  • the fastest response time has been observed on the types of the fourth embodiment (see Fig. 11A , right side). However (as already reported), the droplet 23 will always be sitting on the hydrophobic surface 11 of the working film 10 with a slightly elliptical profile. The second fastest response time has been observed on the types of the third embodiment (see Fig. 11A , left side); the droplet 23 will always be sitting on the hydrophobic surface 11 of the working film 10 with a slightly elliptical profile as well.
  • liquid droplets can be moved in a straight manner on a hydrophobic upper surface 11 of a working film 10 that is placed on an electrode array 20 as depicted in the Figures 10A and 10B , if the flanking electrodes 63 are turned off. This is in good agreement with the observation with respect to embodiment as shown in Fig. 11B , where it has been recognized that the droplets 23 always move in a perfectly straight way towards and from the optically transparent electrodes 60 that are devoid of flanking electrodes 63.
  • the spot (see white spots in the Fig. 11 ) of electrically connecting the optically transparent electrode(s) 60 with the electrode selection unit 64 of the liquid droplet manipulation system 40 is located near the border (e.g. near a corner) of the optically transparent electrode(s) 60 in order to leave as much as possible area for optically investigating the sample in a droplet 23 placed over the optically transparent electrode(s) 60.
  • the spot see white spots in the Figs.
  • the spot (see white spots in the Figs. 10 and 11 ) of electrically connecting the individual electrodes 44 with the electrode selection unit 64 of the liquid droplet manipulation system 40 may be selected as convenient (e.g. in the center of the individual electrodes 44 as shown).
  • the optics of the liquid droplet manipulation system 40 may comprise an optical bottom reading system and a detector connected to the latter. Moreover, the optics of the liquid droplet manipulation system 40 may comprise an optical top reading system and a detector connected to the latter. In addition, the optics of the liquid droplet manipulation system 40 may comprise a combined optical bottom and top reading system and one or more related detectors. These optical systems can be adapted to the inspection or analysis of single droplets 23, but also to the inspection or analysis of larger aggregates of single droplets 23. All optical systems can comprise lenses for influencing the light for exciting or inspection of the samples and also lenses for influencing the light penetrating or emitted by the samples in the liquid droplets 23. With respect to the optics, the arrangement of optical fibers 21 as depicted in the figures 1 to 4 are preferred.
  • all electrodes 44,60,61,63 are located flush with the surface 49 of the substrate 42 or embedded (situated inside, but close to the surface 49) in the substrate 42. All electrical connections preferably are embedded into the substrate 42, at the border of which they can be assembled in a multi-pin plug (e.g. in the case of Fig. 10 ) for connecting the electrode array(s) 20 with the electrode selection unit 64 of the liquid droplet manipulation system 40.
  • the substrate 42 with the electrode array(s) 20 thus can be attached to the central control unit 43 (see Fig. 10 ).
  • the central control unit 43 can be at least partially integrated into the substrate (see Fig. 11 ).
  • the optically transparent electrodes 60 are produced from ITO (Indium tin oxide), which is a solid solution of indium(III) oxide (InzOs) and tin(IV) oxide (SnOz), typically 90% InzOs, 10% SnOz by weight.
  • ITO Indium tin oxide
  • ITO Indium tin oxide
  • InzOs indium(III) oxide
  • SnOz tin(IV) oxide
  • the ITO material is transparent and colorless in thin layers (only in the infrared region of the spectrum, ITO it acts as a metal-like mirror).
  • Indium tin oxide is one of the most widely used transparent conducting oxides because of its two chief properties, its electrical conductivity and optical transparency, as well as the ease with which it can be deposited as a thin film.
  • the electrically insulating film 50, the cover layer 19, the rigid cover 17 are chosen to be optically transparent if required.

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  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Claims (21)

  1. Système (40) de manipulation de gouttelettes liquides, le système de manipulation de gouttelettes liquides (40) comprenant :
    - au moins une cartouche (1) avec un film de travail polymère (10) optiquement transparent pour manipuler dessus des échantillons sous forme de gouttelettes liquides (23) ;
    - un substrat (42) avec au moins un réseau d'électrodes (20) ; et
    - une unité de commande centrale (43) pour commander la sélection d'électrodes individuelles (44) du ou des réseaux d'électrodes (20) et pour fournir aux électrodes (44) des impulsions de tension individuelles pour manipuler des gouttelettes liquides (23) par électromouillage,
    le système de manipulation de gouttelettes liquides (40) étant conçu pour recevoir à la surface des électrodes (44) le film de travail polymère (10) pour manipuler les échantillons sous forme de gouttelettes liquides (23) avec le ou les réseaux d'électrodes (20) lorsque le film de travail polymère (10) est placé sur ledit ou lesdits réseaux d'électrodes (20), et au moins une électrode individuelle sélectionnée (44) du ou des réseaux d'électrodes (20) du système de manipulation de gouttelettes liquides (40) étant conçue pour être pénétrée par la lumière d'un système de détection optique pour l'inspection ou l'analyse optique d'échantillons sous forme de gouttelettes liquides (23) qui sont situés sur le film de travail polymère (10),
    la au moins une électrode individuelle sélectionnée (44) étant conçue sous la forme d'une électrode optiquement transparente (60) ou sous la forme d'une électrode intermédiaire optique (61) qui comprend un trou de passage (62),
    et l'électrode optiquement transparente (60) ou l'électrode intermédiaire optique (61) étant située à la place d'une électrode individuelle (44) sur une trajectoire d'électrode (65) ou dans un réseau d'électrode de type grille (66) et étant conçue pour être manipulée par une unité de sélection d'électrode (64) de l'unité de commande centrale (43),
    dans lequel
    a) l'électrode optiquement transparente (60) est produite à partir d'ITO, ou
    b) un point de connexion électrique entre l'électrode intermédiaire optique (61) et l'unité de sélection d'électrode (64) est situé près d'un angle de l'électrode intermédiaire optique (61).
  2. Système de manipulation de gouttelettes liquides (40) selon la revendication 1, dans lequel l'électrode intermédiaire optique (61) est flanquée d'électrodes adjacentes (63) situées de chaque côté des électrodes intermédiaires optiques (61) et qui sont raccordées à l'unité de sélection d'électrode (64) de l'unité de commande centrale (43).
  3. Système de manipulation de gouttelettes liquides (40) selon la revendication 2, dans lequel les électrodes adjacentes (63) sont de forme triangulaire ou rectangulaire.
  4. Système de manipulation de gouttelettes liquides (40) selon la revendication 2 ou 3, dans lequel les paires opposées d'électrodes adjacentes (63) d'une seule et même électrode intermédiaire optique (61) sont raccordées électriquement entre elles de sorte qu'elles affichent toujours le même potentiel électrique.
  5. Système de manipulation de gouttelettes liquides (40) selon la revendication 1, dans lequel l'électrode intermédiaire optique (61) est divisée en deux électrodes partielles, chacune d'elles comprenant une partie du trou de passage (62).
  6. Système de manipulation de gouttelettes liquides (40) selon la revendication 5, dans lequel les deux électrodes partielles sont raccordées individuellement à l'unité de sélection d'électrode (64) de sorte qu'elles peuvent être activées ou désactivées individuellement.
  7. Système de manipulation de gouttelettes liquides (40) selon la revendication 1, dans lequel l'électrode intermédiaire optique (61) comprend des ailettes latérales qui entourent au moins partiellement une électrode adjacente (44).
  8. Système de manipulation de gouttelettes liquides (40) selon la revendication 1, dans lequel le substrat (42) comprend au moins une fibre optique (21) pour amener de la lumière à une électrode optiquement transparente (60) ou à un trou de passage (62) d'une électrode intermédiaire optique (61), et par conséquent à une gouttelette (23) sur le film de travail polymère (10) qui est placé sur ledit réseau d'électrodes (20).
  9. Système de manipulation de gouttelettes liquides (40) selon la revendication 1, dans lequel le substrat (42) comprend des lignes électriques qui raccordent les électrodes individuelles (44) à une unité de sélection d'électrode (64) de l'unité de commande centrale (43) du système de manipulation de gouttelettes liquides (40).
  10. Système de manipulation de gouttelettes liquides (40) selon la revendication 8, dans lequel la au moins une fibre optique (21) est raccordée à un système de lecture optique inférieur et à un détecteur raccordé à ce dernier, le système de lecture optique inférieur étant conçu pour envoyer une lumière d'excitation à l'échantillon et pour recevoir et détecter la fluorescence émise par un échantillon dans une gouttelette liquide (23) située sur le film de travail polymère (10).
  11. Système de manipulation de gouttelettes liquides (40) selon la revendication 8, dans lequel la au moins une fibre optique (21) est raccordée à un système de lecture optique supérieur et à un détecteur raccordé à ce dernier, le système de lecture optique supérieur étant conçu pour envoyer une lumière d'excitation à l'échantillon et pour recevoir et détecter la fluorescence émise par un échantillon dans une gouttelette liquide (23) située sur le film de travail polymère (10).
  12. Système de manipulation de gouttelettes liquides (40) selon la revendication 8, dans lequel la au moins une fibre optique (21) est raccordée à un système d'excitation optique inférieur et à un système de lecture optique supérieur et à un détecteur raccordé à ce dernier, le système d'excitation optique inférieur étant conçu pour envoyer une lumière d'excitation à un échantillon dans une gouttelette liquide (23) qui est située sur le film de travail polymère (10), et le système de lecture optique supérieur étant conçu pour recevoir et détecter la lumière émise ou transmise par l'échantillon.
  13. Système de manipulation de gouttelettes liquides (40) selon la revendication 8, dans lequel la au moins une fibre optique (21) est raccordée à un système d'excitation optique supérieur et à un système de lecture optique inférieur et à un détecteur raccordé à ce dernier, le système d'excitation optique supérieur étant conçu pour envoyer une lumière d'excitation à un échantillon dans une gouttelette liquide (23) qui est située sur le film de travail polymère (10), et le système de lecture optique inférieur étant conçu pour recevoir et détecter la lumière émise ou transmise par l'échantillon.
  14. Système de manipulation de gouttelettes liquides (40) selon l'une des revendications 1 à 13, dans lequel le film de travail polymère (10) est conçu comme une monocouche d'un matériau hydrophobe.
  15. Système de manipulation de gouttelettes liquides (40) selon l'une des revendications 1 à 13, dans lequel le film de travail polymère (10) est conçu comme un stratifié constitué d'une couche supérieure hydrophobe et d'une couche inférieure diélectrique.
  16. Système de manipulation de gouttelettes liquides (40) selon l'une des revendications 1 à 13, dans lequel une couche, un film ou un revêtement électriquement isolant (50) est appliqué(e) sur une surface (48) des électrodes (44) et sur une surface (49) du substrat (42).
  17. Système de manipulation de gouttelettes liquides (40) selon la revendication 16, dans lequel cette couche, ce film ou ce revêtement électriquement isolant (50) est une couche diélectrique qui recouvre de façon inamovible les électrodes (44) et le substrat (42) du système de manipulation de gouttelettes liquides (40).
  18. Système de manipulation de gouttelettes liquides (40) selon la revendication 16, dans lequel cette couche, ce film ou ce revêtement électriquement isolant (50) est une couche diélectrique supplémentaire qui recouvre de façon amovible les électrodes (44) et le substrat (42) du système (40), la couche, le film ou le revêtement (50) pouvant être remplacé(e).
  19. Système de manipulation de gouttelettes liquides (40) selon l'une des revendications 1 à 18, dans lequel la au moins une cartouche (1) comprend :
    a) un corps (2, 2', 2") qui comprend une surface supérieure (3), une surface inférieure (4) et un certain nombre de puits (5) conçus pour recevoir à l'intérieur des réactifs (6) ou des échantillons (6') ;
    b) une structure supérieure déformable de façon flexible (7) imperméable aux liquides et conçue pour fermer hermétiquement un côté supérieur des puits (5) ;
    c) une structure inférieure perçable (8) imperméable aux liquides et conçue pour fermer hermétiquement un côté inférieur des puits (5) ;
    d) le film de travail polymère (10) étant situé sous la surface inférieure (4) du corps (2, 2', 2") et étant imperméable aux liquides et comprenant une surface supérieure hydrophobe (11) ;
    e) une entretoise périphérique (9, 9', 9") située sous la surface inférieure (4) du corps (2, 2', 2") et raccordant le fim de travail polymère (10) au corps (2, 2', 2") ;
    f) un espace (12) entre la surface inférieure (4) du corps (2, 2', 2") et la surface supérieure hydrophobe (11) du film de travail polymère (10), l'espace (12) étant défini par l'entretoise périphérique (9, 9', 9") ; et
    g) un certain nombre d'éléments de perçage (13) situés sous la structure inférieure perçable (8) et configurés pour percer les structures inférieures perçables (8) pour libérer les réactifs ou les échantillons (6, 6') des puits (5) dans l'espace (12).
  20. Système de manipulation de gouttelettes liquides (40) selon la revendication 19, dans lequel le film de travail polymère (10) de la cartouche (1) est conçu sous la forme d'une monocouche d'un matériau électriquement non conducteur, la surface supérieure (11) du film de travail polymère (10) étant traitée pour être hydrophobe.
  21. Système de manipulation de gouttelettes liquides (40) selon la revendication 19, dans lequel la cartouche (1) comprend au moins une fibre optique (21) pour amener de la lumière à une gouttelette (23) dans l'espace (12) et/ou pour éloigner la lumière d'une gouttelette (23) dans l'espace (12).
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CN107649223B (zh) * 2017-09-27 2019-10-15 京东方科技集团股份有限公司 液滴控制检测器件及其工作方法
CN113101985B (zh) * 2019-06-26 2022-07-22 京东方科技集团股份有限公司 检测芯片和检测系统
CN114173925A (zh) 2019-06-26 2022-03-11 帝肯贸易股份公司 药筒、电润湿样品处理系统及其进料

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