WO2007115815A1

WO2007115815A1 - Method and apparatus for labelling, reading and sorting of microcarriers

Info

Publication number: WO2007115815A1
Application number: PCT/EP2007/003168
Authority: WO
Inventors: Christiaan Roelant; Marc Leblans; Philip Van Donink
Original assignee: Pauwels, Rudi
Priority date: 2006-04-07
Filing date: 2007-04-10
Publication date: 2007-10-18

Abstract

The invention provides microfluidic devices, for the manipulation of magnetic microcarriers, comprising means for generating at least one magnetically and electrically defined cage within a microfluidic container for positioning and orienting of the magnetic microcarriers. The invention further provides microfluidic containers for use in the devices of the invention and methods for the identification and/or encoding of magnetic microcarriers which comprise positioning and orienting the microcarriers in an electrically and magnetically defined cage.

Description

Method and apparatus for labelling, reading and sorting of microcarriers.

FIELD OF THE INVENTION The invention relates to methods of manipulation and orientation of microcarriers in a device using magnetic and electric fields, and apparatus therefor.

BACKGROUND Fluidic systems offer an intrinsic compatibility with microcarrier-surface- based assays (immuno-assays, DNA-based assays) because of reduced reagent consumption, fast reaction times and large surface-to-voiume ratios. For this reason, microfluidic systems are becoming the assay recipient of choice for the processing of microcarriers. The processing of cells or beads using combinations of dielectrophoretic microelectrodes has been described in e.g. WO0470362 and Mϋller et al. (2003) IEEE Eng. Med Biol. Mag. 22, 51-61. This system is particularly advantageous for the sorting of cells, as the dielectrophoretic handling allows recovery of the cells without damage. These devices envisage sorting of the cells based on the (e.g. fluorescent) signal generated by the cells or beads as a result of a previous manipulation thereof, resulting in the presence of a selectable label (e.g. transfection, immunological reaction). Thus, in view of the finite number of labels available and the intrinsic difficulties of screening based on label intensity, these systems are not ideal for sorting in the context of high- throughput or multiplexing assays.

Encoded microcarriers and their advantages are reviewed for example in

Braeckmans et al. (2002) Nat Rev Drug Discov. 1, 447-456. In order to allow detection of the code, microcarriers have been developed which allow orientation of encoded microcarriers using a magnetic field (Braeckmans et al.

(2003) Nat. Mater. 2, 169-173 and WO02/33419). The ability to ensure an integrated individualised but high-throughput processing of encoded microcarriers would make it possible to add another dimension to multiplexing, while at the same time providing a tool for improving the accuracy of the identification and/or encoding procedure of the beads.

SUMMARY OF THE INVENTION

The object of the invention is to provide methods and devices for multiplex analysis using microcarriers.

In a first aspect the invention provides devices for the manipulation of magnetic microcarriers in a liquid, characterised in that the devices comprise means for generating at least one magnetically and electrically defined cage within a microfluidic container for positioning and orienting magnetic microcarriers.

According to the invention, the magnetically and electrically defined cage is provided by a means for generating an electric field, such as dielectrophoretic electrodes, and a means for generating a magnetic field.

According to a particular embodiment, the microfluidic container is an integrated part of the device of the invention. Alternatively, the microfluidic container is a removable, optionally disposable, container, which is placed within the device of the invention. The means for generating an electric field and for generating a magnetic field can be provided as an integrated part of the microfluidic container or can be provided within the device for application to the microfluidic container. According to one embodiment, the microfluidic container is provided with dielectrophoretic electrodes which can be used to generate an electric field within the microfluidic container when attached to a suitable power source.

According to a further embodiment, the devices of the present invention further comprise a detection module, which allows detection of the microcarriers when trapped in the magnetically and electrically defined cage. Alternatively, the devices of the present invention are provided for use in combination with an independent detection module. According to the present invention, the integrated or separate detection module allows for (simultaneous) detection of both a microcarrier signal and/or a marker signal generated by a microcarrier. According to one embodiment, the detection of the microcarrier signal is performed with the same detection device as the detection of the marker signal. In a particular embodiment, the detection device is a confocal or pseudo- confocal microscope, most particularly a fluorescence microscope. Alternatively, two different detection devices can be used.

According to one embodiment, the marker signal generated by the microcarrier is indicative of a reaction which has taken place on the surface of the microcarrier. Optionally, this reaction involves a target-probe, provided at the surface of the microcarrier, which is capable of binding to a target.

Further embodiments of the invention relate to the devices described above which, in addition, comprise integrated means for sorting the microcarriers based on their microcarrier and/or marker signal.

Yet another embodiment of the invention provides devices which further comprise an encoding device, capable of encoding a microcarrier signal on a magnetic microcarrier when trapped in the electrically and magnetically defined cage. Most particularly, the encoding device is a high spatial resolution light source, capable of bleaching a bleachable substrate.

In another aspect, the invention provides methods for the identification or encoding of a magnetic microcarrier, which comprise positioning and orienting the microcarrier in a liquid in an electrically and magnetically defined cage by application of an electrical and magnetic field, so as to ensure a specific position and orientation of the microcarriers for detection, identification and/or encoding.

In one embodiment, the method comprises one or more detection steps with the microcarrier trapped in the electrically and magnetically defined cage, whereby the detection step is performed by use of any suitable optical or optoelectronic device, e.g. a confocal microscope. Most particularly, the detection module used for detection is placed so that its direction of detection (e.g. optical axis) is perpendicular to the magnetic field.

Typically, the detection steps comprise the detection of a microcarrier signal, for the identification of the microcarrier, and/or the detection of a marker signal, e.g. for the determination and/or quantification of a reaction that has taken place at the surface of the microcarrier.

In a further embodiment, the method of the invention further comprises a sorting step, whereby sorting is performed based on the detection of the marker and/or microcarrier signal.

In a further embodiment, the method comprises an encoding step and the encoding is ensured by bleaching a pattern on a bleachable substrate, such as by a high spatial resolution light source.

The present invention relates in one embodiment to the combined use of a microfluidic container, an optical or opto-electronic detection device, e.g. a pseudoconfocal microscope, dielectric electrodes and a magnetic field generator.

This invention focuses on the combined use of an exchangeable 3D microsystem (such as microfluidic chips (with or without integrated micro- electrode systems), photo-conductive cells and lab-on-chip structures) enabling the manipulation of individual microcarriers (or multiple microcarriers in parallel) which can be used in the encoding of the microcarriers (such as by laser- assisted writing of an optically directly readable code inside said microcarriers) and in the identification and sorting of the microcarriers based on their code.

DETAILED DESCRIPTION OF THE INVENTION

definitions

A "fluidic device" as used herein refers to a device wherein particles are transported in fluid. Typically a fluidic device comprises channels through which the particles, such as microcarriers, are moved by the current of the flow and/or other forces such as electric fields generated within the fluidic device. The term "microcarrier" refers to a particle with a diameter between about

1 μm and 1000 μm (micrometer), the surface of which is suitable for binding probes. Synonyms include (micro)particle or (micro)bead or (micro)sphere.

The term "target" or "analyte" as used herein refers to the molecule to be detected or identified in the detection methods of the present invention. The term "target-probe" as used herein, refers to a probe capable of (specifically) detecting the target of interest. Typical target-probes include oligonucleotides complementary to the target DNA, antibodies capable of binding to the target protein or polypeptide, but can include proteins, ligands, specific polymeric surfaces, etc.

The term "marker signal", as used herein, refers to the signal, or a change in the signal, emitted by a label, that can be attached to a probe or a target-probe or to a molecule capable of binding the target-probe or the target according to the invention. Where the label is a fluorescent molecule, the term "marker fluorescence" is also used. Detection of the marker signal can be indicative of the outcome of the analysis/reaction performed on the surface of the microcarrier in the methods of the present invention.

"Microcarrier signal" refers to the signal, or a change in the signal, that is used in the identification of the microcarrier, which allows the reading of a code provided on the microcarrier. Where the microcarrier signal is based on fluorescence, the term "microcarrier fluorescence" is also used.

In the context of the present invention, "a code" refers to a spatial modulation created inside the microcarrier or on its outer surface. This spatial modulation may be defined as a known arrangement of a finite number of distinct volume elements located inside or on the surface of the microcarrier. The known arrangement of distinct volume elements can be generated by (i) changing one or more properties of the material in an individual volume element, or (ii) by removing material from an individual volume element, or (iii) by depositing material on an individual volume element, or (iv) by leaving an individual volume element unchanged, or a combination of the above possibilities. This known arrangement for example, may be such that these volume elements lie on one or more dimensions such as on a line arrangement or in a plane. Any reference in this application to codes written "on" the microcarriers thus includes codes written on the surface of the microcarriers as well as codes written at an internal depth of the microcarriers. Preferred codes are optically readable codes.

An "electrically and magnetically defined cage" as used herein refers to a combination of electric and magnetic fields positioned in such a way that it ensures positioning of a magnetic microcarrier in a fluid (using the electric field) and allows specific orientation thereof (using the magnetic field).

A "microfluidic container" as used herein refers to a container in which fluid can be contained, which is essentially closed off, with the exception of input and output openings and which comprises at least one area on its surface which is transparent, to allow detection, with a detection device, of a microcarrier located in the container.

The present invention relates to methods and devices for manipulating encoded microcarriers in a fluidic environment. The invention is based on the surprising observation that magnetically coated beads can be manipulated in an electric field without interference by their magnetic properties.

Compared to the existing fluidic devices wherein the manipulation of objects using an electric field has been described, the present invention provides an additional level of manipulation based on the generation of a magnetic field. This is of particular interest in the manipulation of encoded microcarriers. Indeed, using the state of the art devices, it may be possible to immobilise and observe a microcarrier (e.g. through a microscope). However, identification of a (e.g. asymmetrical) code on the microcarrier in addition requires the proper orientation of the microcarrier. Orientation of the microcarrier can be achieved by using magnetically coated microcarriers and subjecting them to a magnetic field upon identification. Surprisingly, it has been found that the caging via an electrical field is not distorted by the presence of metallic material in the microcarrier or by the application of a magnetic field on such a particle. In addition there is no interference of the electrical field on the rotating of a microcarrier using magnetic forces. This not only allows accurate manipulation and identification of microcarriers in a fluidic device but moreover allows integrated signal-based manipulation of the microcarriers. Finally, it provides methods and devices for digitally accurate automated encoding of microcarriers.

In a first aspect, the invention relates to a device which comprises, a means for generating at least one electrically and magnetically defined "cage" or volume for capturing and orienting a microcarrier. According to one embodiment, the device (Figure 2) comprises a microfluidic container (2), wherein, upon applying an electric field and a magnetic field, at least one electrically and magnetically defined "cage" is generated (1 ). The means for applying an electric field and/or the means for applying a magnetic field can be integrated in the microfluidic container or can be external thereto. Optionally, the means for applying an electric field are electrodes integrated into the microfluidic container.

According to a particular embodiment the device further comprises a detection module and/or an encoding module.

The microfluidic container thus can be a part of an integrated device or can be a separate, optionally disposable component, in which the electrically and magnetically defined "cage" can be generated by application of an electric and magnetic field, and which can be used in combination with external or independent detection and/or encoding modules. Typically the detection module comprises an optical platform (4) and a microscope (5) with a pseudo-confocal unit (6).

According to one embodiment, the device further comprises, either integrated therein, or as part of the microfluidic container, channels and/or compartments which guide the microcarriers of the invention to and from the "cage" and optionally through different "reaction chambers", wherein reactions can take place on the surface of the microcarriers. According to a specific embodiment, one or more of the channels and/or compartments is provided with electrodes (7) which, by repulsion direct the microcarriers through the device. Additionally or alternatively, the device further contains one or more integrated sorting devices such as valves (8). In one embodiment, the sorting is achieved by one or more electrodes. The device can further comprise additional elements, such as but not limited to, connections to multiple sample recipients into which microcarriers can be sorted, a CCD camera, and a microcarrier reservoir.

The means for applying an electric field envisaged in the context of the present invention are means which allow the manipulation of a microcarrier in the microfluidic container. Typically, the means for applying an electric field are integrated within the microfluidic container, and the container can be connected to an electrical power source. Alternatively however, it is envisaged that the means for applying an electric field are provided as a separate module for application to the surfaces of a microfluidic container, which can be disposable.

The means for providing an electric field are used in the devices and methods of the present invention to create a cage, more particularly a dielectric field cage (DFC) that allows long-term stable caging of a microcarrier in a defined position. A DFC typically consists of eight electrodes arranged symmetrically. It can be driven with different phase patterns. The two most commonly used are "rotating" and "alternating". In both cases the electric field vanishes in the centre of the cage (trapping point). A rotating drive creates a closed dipole force potential. This ensures both trapping of enclosed objects and efficient repelling of outside microcarriers. Although rotating electric fields are applied, no rotation of objects is induced in the central vertical plane. To rotate a caged microcarrier, symmetry of driving must be broken. The alternating mode is counter-intuitive because upper and lower electrode planes are driven with the same phase pattern. Nevertheless, once trapped, forces against flow are high. As dipole trapping force is zero in the central vertical axis, objects much smaller than the cage dimension cannot be levitated.

Further manipulations of the microcarriers typically envisaged in the context of the device and methods of the present invention using an electrical field, is the movement and/or sorting of the microcarriers within the microfluidic container. Typically, this is ensured by way of specifically oriented electrodes. Zigzag or hook arrangements are used as parking or holding elements against the flow. A deflector can be provided that allows the separation of objects according to their size and/or dielectric properties using the equilibrium between DEP force and flow component normal to the electrode pair. Flow rates ranging from 10 μm/s up to several mm/s are described. This is achieved by angling the electrodes against streaming. A deflector is used to guide microcarriers into other channels for subsequent loading, separation and/or washing. Several deflectors can be combined behind one another or side by side to create a so- called funnel, to ensure the guiding of the microcarrier on a well-defined trajectory. Typically, a funnel will guide the microcarrier to the DFC.

US6727451 describes a three-dimensional electrode configuration which generates an electrical field barrier which allows the guided movement of a microcarrier. Other suitable methods to manipulate microcarriers with electrical fields in accordance with the present invention include the movement of microcarriers by DiElectroPhoresis (DEP) and the retention of microcarriers by caging them in an electrical field. Devices for manipulating individual microcarriers using an electrical field are described in the art, for example in Mϋller et al. (2003, IEEE Eng. Med Biol. Mag. 22, 51-61 ) which describes configurations using microelectrodes for the movement and caging of objects, which can be applied to the microcarriers according to the present invention. WO2004070362 describes a configuration as described above for isolating cells with a fluorescent label from a cell population.

The device of the present invention further comprise one or more means for generating a magnetic field. Most particularly, the magnetic field is applied to provide the electrically and magnetically defined cage(s), so as to ensure the orientation of a microcarrier once it has been trapped within the electrical field. The means for applying a magnetic field can be integrated in the microfluidic container or can be applied thereto when appropriate. Means for applying a magnetic field are known in the art and include e.g. electromagnetic coils and permanent magnets. More particularly, methods of applying such means for orienting magnetic microcarriers are described in WO0233419. According to a particular embodiment, the magnetic field is applied to the microcarrier trapped in the "cage" at an angle which is perpendicular (90°) to the direction of detection (see below). A further particular embodiment of the present invention relates to a device wherein the magnetic field is applied perpendicularly both to the detection direction and perpendicular to the direction in which the code is written on the bead.

The strength of the magnetic field applied to the "cage" in the context of the present invention is determined by a) the strength required to allow orientation of the microcarrier and b) the strength applied to generate the code on the microcarrier. Generally, the strength of the magnetic field applied to the microcarrier in the device of the present invention should not exceed the strength of the magnetic field used to write the code onto the microcarrier (see below) so as not to change the magnetic orientation of the microcarrier. A device of the present invention thus comprises a microfluidic container comprising an electrically and magnetically defined "cage" to/from which the microcarriers are guided and wherein the individual microcarriers can be trapped for detection and/or encoding purposes. Moreover, the magnetic field applied to the "cage" further allows orientation of the microcarriers for detection and/or encoding of a microcarrier signal.

According to a particular embodiment, the device of the invention comprises a detection module. Alternatively, the device of the present invention can be used in combination with a suitable detection module. The detection module ensures the detection of the microcarrier signal and/or the marker signal and thus the nature of the detection module is determined by the nature of the marker signal and/or microcarrier signal. Where one or both of the signals are optical, detection and identification can be carried out with an optical microscope. The detection of different types of microcarrier signals is envisaged within the context of the present invention. According to one embodiment, the microcarrier signal is a result of the radiation emitted by the core of the microcarrier, which, by contrast allows the reading of a code applied thereon. According to a particular embodiment, the microcarriers are encoded using the methods described in WO063695. In this embodiment, the microcarriers contain a bleachable substance, and the codes on the microcarriers are in the form of bleached patterns within the bleachable portions of the microcarriers. The microcarriers contain the bleachable substance either on the surface of the core of the microcarrier or within the core of the microcarrier. The bleachable substance can be mixed with the core material upon generation of the core particles of the microcarrier or can be applied to the core of the microcarrier as a separate layer, optionally by specifically linking bieachable molecules to the surface of the core materia!. Bleachable substances particularly envisaged within the context of the invention include bleachable fluorescent or electromagnetic radiation absorbing substances. Typically bleachable luminophores can be used. Examples of luminophores include fluorescers, phosphorescers, or scintillators. Bleachable chemiluminescent, bioluminescent, or colored substances may be used. The bleachable substances may be, more specifically, fluorescein isothiocyanate ("FITC"), phycoerythrines, coumarins, lucifer yellow, and rhodamine. Alternative embodiments of the bleachable substances include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Acridine Orange, Acridine Red, Acridine Yellow, Acriflavin, AFA (Acriflavin Feulgen SITSA), Alizarin Complexon, Alizarin Red, Allophycocyanin, ACMA, Aminoactinomycin D, Aminocoumarin, Anthroyl Stearate, Aryl-or Heteroaryl-substituted Polyolefin, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, BOBO 1 , Blancophor FFG Solution, Blancophor SV, BodipyFI, BOPRO1 , Brilliant Sulphoflavin FF, Calcium Blue, Calcium Green, 99.7%. The bleachable substances should be chosen so that, when bleaching occurs, the code remains on the microcarrier for the period of time that is desired for the use of the microcarriers and any necessary reading of the codes. Thus, a certain amount of diffusion of non-bleached molecules into the bleached areas is acceptable as long as the useful life of the code is preserved.

As indicated above, the detection module of the device of the present invention can additionally or alternatively detect a marker signal on the microcarrier, which provides information on a reaction that has taken place on the surface of the microcarrier. Typical reactions envisaged in the context of the present invention are chemical or biological reactions, such as, but not limited to antibody/antigen, antibody/hapten, receptor(fragment)/ligand, sugar/lectin, complementary nucleic acid binding (RNA or DNA, or combination thereof), enzyme/substrate, enzyme/cofactor, enzyme/inhibitor, protein/aptamer etc. The marker signal is generated by the direct or indirect binding of a label to the microcarrier. Typically, the marker signal is generated by the indirect binding of a labelled target-probe to the microcarrier, and the marker signal is indicative of the presence and/or amount of target bound to the microcarrier (and thus present in a sample). Alternatively, the marker signal can be the quenching of a signal previously provided on the microcarrier, e.g. as the result of the binding of a compound to a labelled microcarrier. The marker signal can be the result of a label which is directly detectable (based on its inherent properties, e.g. radiation) or can be dependent on specific conditions (such as presence of substrate for an enzyme label, etc.). Typical labels useful in the context of the present invention are those labels which are classically used in in vitro assays such as, but not limited to, chromophoric groups, radioactive labels, electroluminescent, chemiluminescent, phosphorescent, fluorescent or reflecting labels. The invention further provides for the simultaneous detection of different marker signals, whereby the different marker signals can e.g. be chromophoric groups with differing emission spectra, such as series of different fluorescent dyes, quantum dots, and other luminescent material, such as nanophosphor.

Typically, one or more microcarrier signals and/or marker signals are fluorescent and the detection device suitable for reading these one or more microcarrier signals and/or marker signals is a fluorescence confocal microscope, as it allows rejection of out-of-focus interference. Examples of confocal microscopes include those that have laser excitation and the capability to simultaneously detect two or more emitted colours. Standard excitation sources are the argon ion laser that emits at 488 nm and 514 nm and the argon/krypton mixed gas laser that gives three useful spectral lines for excitation at 488 nm, 568 nm and 647 nm. These wavelengths cover many of the commonly used fluorophores. DNA stains DAPI and Hoechst 33258 require UV excitation, which can be ensured e.g. by large-frame, high-power argon ion lasers. Either point-scanners or multiple-beam confocal microscopes can be used. Most particularly, a pseudoconfocal microscope is used (such as, but not limited to spinning disk (hole illumination, line illumination, or slit illumination) detection. According to a particular embodiment of the present invention, a pseudo-confoca! or structured elumination microscope with a (double) Nipkow disk is used. When radioactive labels are used, detection can also occur using a gamma counter, scintillation counter or (phosphor)imaging device. Suitable detection devices for use in the detection of labels such as chromophoric groups, radioactive labels, electroluminescent, chemiluminescent, phosphorescent, fluorescent or reflecting labels and others are known in the art.

According to a further aspect the device and methods of the invention further comprises an encoding module or an encoding step. The encoding module can be combined with the detection module or can be adjacent thereto. The nature of the encoding device present in the encoding module will be determined by the nature of the code used to identify the microcarriers. Suitable encoding devices, appropriate for different types of codes, are known to the skilled person, a few examples of which are provided below. Typically, according to the present invention, encoding of a microcarrier is ensured upon trapping the microcarrier in a magnetically and electrically defined cage as described herein, whereby the microcarrier is oriented by the application of a magnetic field. The application of this same magnetic field to the magnetically and electrically defined cage during detection will ensure that the microcarrier is adequately oriented to allow the reading of the code. Where the code is a bleached pattern on a bleachable substrate, the encoding module used in the methods and devices described herein can be a high spatial resolution light source, such as a laser, a lamp, or a source that emits X-rays, alpha and beta rays, ion beams, or any form of electromagnetic radiation. Alternatively, the encoding device can be a photochroming or chemical etching device. According to a particular embodiment, the encoding module comprises a high spatial resolution light source, and in particular a laser or a lamp, as described in WO0063695. Barcodes can be written by means of the methods described in WO0063695, by using a cylindrical lens such that the beam is focussed to a line perpendicular to the scanning direction, instead of being focussed to a spot. The focussing of the beam to a line requires significantly higher laser power, in order to obtain the same bleaching level. Alternatively 'dotcodes' are obtained by using a spot focus, which reduces the 'height' of the barcode to the spot size. Depending on the method used, different encoded sites are envisaged on the microcarrier within the context of the present invention. According to a particular embodiment of the invention, the microcarriers are provided with a code at an internal depth of the microcarriers, more particularly in the centre plane of the microcarriers. Depending on the shape of the microcarrier, writing the code on the centre plane can be advantageous as it provides the largest surface area available for writing. Furthermore, for microcarriers having curved surfaces, writing the code on the centre plane is advantageous in that the flat plane facilitates reading and or writing compared to the curved surface.

The codes of the present invention may be of any geometry, design, or symbol that can be written and read on the microcarriers. Typically according to the present invention, the code is written in a specific orientation on the microcarrier and the reading of the code requires ensuring that same orientation of the microcarrier. For example, the codes may be written as numbers or letters, or as codes in the form of symbols, pictures, bar codes or ring codes. Ring codes are similar to bar codes, except that concentric circles are used rather than straight lines. A ring may contain, for example, the same information as one bar. According to a particular embodiment of the invention, the codes are one- or two-dimensional, in which information is stored along one or two spatial directions only (dot or barcodes).

Another aspect of the invention relates to a method for the manipulation of microcarriers suspended in a liquid using a combination of magnetic and electrical fields. According to a particular embodiment, the method comprises trapping the microcarrier in an electrically and magnetically defined cage, which allows detection of one or more signals generated by the microcarrier by a detection module. The signal of the microcarrier most particularly comprises the microcarrier signal(s), which allows the identification of the microcarrier based on its code. Optionally, the microcarrier also generates one or more marker signal(s), which allow(s) the reading of the result of a reaction which has taken place at the surface of the microcarrier. Using the method of the present invention, microcarriers can not only be held in a fixed position before the detection module but also oriented such that the code which is present on the microcarrier can be read.

Additionally or alternatively, the manipulation of the microcarrier comprises the trapping the microcarrier in an electrically and magnetically defined cage which is oriented such that the provision of one or more codes can be ensured on the microcarrier by an encoding module. Here too, the specific individual positioning and orientation of each microcarrier within the electrically and magnetically defined cage ensures accurate encoding and the ability to ensure accurate read-out (when that same magnetic field is applied for detection).

Thus the present invention provides methods for manipulating microcarriers in a fluidic device which allows optimal manipulation of microcarriers to allow precise reading and/or writing of codes on the microcarriers.

Optionally, the manipulation further comprises one or more steps ensuring the movement of the magnetic microcarrier by way of an electrical field to and from the magnetically and electrically defined cage(s) within the devices of the present invention. Additionally or alternatively, the manipulation comprises the selection of the microcarrier based on the marker and/or microcarrier signal detected by the detection module and the specific movement thereof based thereon. Thus, one embodiment provides for the manipulation of the microcarrier as determined by the detection of the microcarrier and/or marker signal by the detection module. Based on the marker and/or microcarrier signal detected, a different electric field is applied to deviate the microcarrier into a certain direction for further trafficking and/or isolation.

The methods and devices of the present invention involve the use of magnetic microcarriers. Typically the microcarrier will comprise a core and a coating. The core or central part of the microcarrier is a reaction volume or a support which may be produced from any material that is routinely employed in high-throughput screening technology and diagnostics. For example, the core of microcarriers may be made from a solid, a semi-solid, or a combination of a solid and a semi-solid material, and can be a support such as used in chemical and biological assays and/or chemical synthesis. Non-limiting examples of these materials include cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, agar, pore-glass, silica gel, polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, polysulfone, grafted copolymer such as polyethylene glycol/polystyrene, cross-linked dextrans, methylstyrene, polypropylene, acrylic polymer, paramagnetic, carbon, graphite, polycarbonate, polypeptide, hydrogels, liposomes, proteinaceous polymer, titanium dioxide, latex, resin, lipid, ceramic, charcoal, metal, bentonite, kaolinite, rubber, polyacrylamide, latex, silicone, e.g., polydimethyldiphenyl siloxane, dimethylacrylamide, and the like or combinations thereof.

According to a particular embodiment, the core of the microcarrier is itself a microparticle, i.e. a solid or semi-solid particle. Most particularly, the microparticle used for the microcarrier core is made of latex, polystyrene, or cross-linked dextrans.

The size and shape of the microcarrier are not critical to the present invention. Most particularly, the microcarriers of the present invention are of a shape and size that is suitable for encoding, positioning, orienting and detection thereof. According to a particular embodiment of the invention, the microcarriers are in the form of spheres. According to a particular embodiment the diameter of the microcarriers used in the context of the present invention is between 10 μm and 500 μm, more particularly between 20 μm and 100 μm. The microcarriers used in the methods and devices according to the present invention are furthermore characterised by the fact that they are magnetic. According to one embodiment, the microcarriers are magnetised by providing, on top of the core of the microcarriers, a coating of magnetic particles. The term "magnetic" as used herein includes all types of material that respond to a magnetic field, such as, but not limited to ferromagnetic, paramagnetic, and supermagnetic materials. According to a particular embodiment of the present invention, the magnetic particles are nanoparticles, more particularly ferromagnetic particles. Examples of ferromagnetic particles include, but are not limited to metal oxide particles, such as but not limited to chromium oxide, or ferric oxide particles. Particular examples of magnetic particles include particles of C^O₃, Fe₂θ₃, Fe₃O₄, Ni-and Co-metals, other metal oxides and metals. The ferromagnetic material can be provided on the microcarrier at a concentration ranging from 0.1 to 50 % by weight, or at a concentration ranging from 0.5 to 40 %, or for example at a concentration ranging from 1 to 20% of the total weight of the microcarrier.

According to an embodiment of the present invention, the size of the magnetic particles is between 50 nm and 500 nm, more particularly between 100 and 400 nm. Magnetic particles suitable for use in the coating of the present invention are available commercially (such as, but not limited to, from Sigma-Aldrich). Optionally, such stock solutions are ultrasonicated as to keep particles unaggregated in suspension.

Magnetic particles can be obtained by producing a metal oxide stock solution with an average size of less than 500 nm. This can be done by heating and precipitating a mixture of divalent and trivalent metal salts in a ratio 3/2 (as described in WO9109141). The solution of magnetic particles can optionally be filtered through a 0.1 μm, 0.22 μm, or 0.45 μm pore filter.

The magnetic microcarriers used in the context of the present invention can optionally comprise one or more probes, which are envisioned to react with a target. The targets and probes envisaged in the methods of the invention generally include but are not limited to proteins, peptides, DNA, RNA, and chemical molecules. A non-exhaustive list of the envisaged embodiment of the targets and probes according to the present invention include antibodies or any fragments thereof, receptors or receptor ligands, carbohydrates, small molecules, enzymes or enzymattcaliy active fragments thereof, enzyme inhibitors, enzyme substrates, DNA probes, RNA probes, bacteria! and viral antigens, amino acids, molecules from chemical libraries, peptide libraries, DNA libraries, pharmaceutical compounds etc.

The microcarriers of the present invention can be applied in a variety of analyses and reactions, which can optionally take place within the microfluidic device of the present invention, most particularly within one or more 'reaction chambers' provided in the microfluidic container. Typical analyses are qualitative and/or quantitative detections of analytes, such as, but not limited to infectious agents and toxins in biological or environmental samples, contaminants in food or feed products, by-products of chemical reactions, etc. Exemplary qualitative and/or quantitative reactions are performed using target specific probes which can be provided at the surface of the microcarriers, as described above. The detection of the reaction can be ensured in different ways. Commonly, this is ensured by labelling the captured analyte, either prior to its binding to the target-probe (e.g. incorporation of a label in the PCR product of a DNA analyte) or after its binding to the target-probe (by use of a second target-specific probe which is labelled).

A particular embodiment of the present invention provides for a device which comprises a means for generating an electrically and magnetically defined cage and methods for using such a device in multiplexing assays. Typically the device of the invention comprises, or is used in combination with, a microfluidic container, in which magnetic coded microcarriers such as described in WO0233419 (fluorescent magnetic polystyrene microcarriers of 40 μm diameter containing a probe) are loaded. More particularly, in the device of the present invention, different reaction chambers are created within the microfluidic container by way of electrodes and the microcarriers are guided through these microfluidic chambers using (di)electric fields that are generated by the electrodes. Most particularly the microfluidic container is a mechanical barrier- free device. The device and methods of the invention are characterised in that, subsequent to the reaction chamber(s) (wherein optionally different reactions have taken place with the probe present on the surface of the microcarrier), the microcarrier is guided to an eiectricaliy and magnetically defined cage, which is aligned with a detection module or unit. The microcarrier is trapped in the cage by an electrical field, and oriented using a magnetic field, so as to allow detection of the microcarrier signal to read the code. The detection module comprises a transparent surface or optical platform on which the microfluidic container is placed and through which the caged microcarrier can be detected by the detection device. In a particular embodiment, both the marker signal (which is present on the microcarrier as a result of the reaction in the reaction chamber) and the microcarrier signal (i.e. the code present on the microcarrier) are detected by the same detection device, such as by a microscope, by manipulating the focus of the objective. The information of the microcarrier signal is optionally processed electronically and can be used to direct valves within the fluidic device, allowing sorting of the microcarriers. Alternatively, the microcarrier signal and/or marker signal is used to direct the collector, which ensures that the microcarriers are differentially collected, depending on their signal (see also prior art device in Figure 1 ).

Surprisingly, processing of the preferred microcarriers by applying an electric field barrier, is not influenced by the fact that these microcarriers are magnetic. Furthermore, while they are entrapped into an electric field "cage" the magnetic microcarriers can still be turned around and oriented for reading of their code. If no code is present or a further code is needed, such a code can be written on an entrapped microcarrier, in the presence of a magnetic field. After reading or writing of a code, based on the code that is read or written, an instruction can be given for further processing of the entrapped microcarrier into a specific direction or recipient.

BRIEF DESCRIPTION OF THE FIGURES

The following Examples, not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which: Figure 1 : Prior art configuration microfluidic device comprising a pseudoconfocal module (1 ) linked to a microscope unit ( 3 ) with a laser beam entry port (2) microscope objectives (4) (5), 3D- microstructure (7 ) holder device (6) , and connection to a sample recipient into which microparticles can be sorted. CCD camera

(10) and microparticle reservoir ( 9 ).

Figure 2: Microfluidic set up according to one embodiment of the invention comprising a microfluidic container (2) in which an electrically and magnetically defined cage (1) can be generated (by a combination of electrodes and a magnetic field generator (3)), a detection module comprising an optical platform (4), a microscope (5) provided with a pseudo-confocal unit (6), one or more integrated sorting valves (8), and specifically oriented electrodes (7).

EXAMPLES

Example 1 : A microfluidic device according to the invention.

One embodiment of the microfluidic sorting system according to the invention is described in Figure 2. Briefly, the set-up comprises a microfluidic channelling system with a detection module comprising an inverted optical platform (4), a microscope (5) with a pseudo-confocal unit (6), a magnetic field generator (3), one or more sorting valves (8) and an electrical cage (1).

The device of the invention can further comprise integrated pneumatic control lines for pumps and valves.

Basically, magnetic microparticles are transported through the microfluidic device by the current of the flow and dielectrophoretic guidance as described in WO2004/070362. Using dielectrophoretic electrode funnel-shaped arrangements (7), the particles can be guided through the microfluidic channels sequentially. The particles can be held in the detection module using a dielectrophoretic cage (1 ), which can be switched on and off. Once placed in the detection cage, the microparticles are oriented by application of the magnetic field, and the code (microcarrier signal) is read through the inverted optical platform by a pseudoconfocal microscope. The marker signal of the bead can be read using the same pseudoconfocal microscope. The sorting of the microparticles by the valves (8) can be guided by either the marker and/or the microcarrier signal.

Example 2: Manipulation of magnetic microcarriers caged in an electric field

Encoded Fluorescent magnetic microcarriers of 40 μm were suspended in normo-osmotic sugar solution. The microcarriers were introduced into the microfluidic device of example 1. A microcarrier was caged between the electrodes by applying a voltage in the range of 1-100 Volts. Typically the microcarrier is caged for about 0.1-1 second. Subsequently a magnetic field of tenfolds of Gauss (10OkHz-IMHz) was applied in order to orient the microcarrier, while maintaining the microcarrier in the electric cage. The positioned microcarrier is illuminated and the emitting fluorescence is recorded by the confocal microscope. The code on the microcarrier was read by changing the focus of the confocal microscope.

The information of the code is used to direct the valves so as to ensure the appropriate sorting of the caged microcarrier and collection of the carrier in a specific recipient.

Claims

1. A device for the manipulation of magnetic microcarriers in a liquid characterised in that the device comprises means for generating at least one magnetically and electrically defined cage within a microfluidic container for positioning and orienting the magnetic microcarriers.

2. The device according to claim 1 , wherein said electrically defined cage is provided by dielectrophoretic electrodes.

3. The device according to claim 2, wherein said dielectrophoretic electrodes are provided within said microfluidic container.

4. The device according to any one of claims 1 to 3, for use with microcarriers having a microcarrier signal, wherein said device further comprises a detection module allowing the detection of a microcarrier signal.

5. The device according to claim 4, for use with microcarriers having a target- probe capable of generating a marker signal, wherein said device further comprises a detection device capable of detecting a marker signal.

6. The device according to claim 5, wherein said detection device capable of detecting a marker signal is also capable of detecting a microcarrier signal.

7. The device of claim 6, wherein said detection device is a confocal microscope.

8. The device according to claim 7, wherein said microscope is a fluorescence microscope.

9. The device according to any one of claims 1 to 8, further comprising integrated means for sorting microcarriers based on a microcarrier and/or a marker signal.

10. The device according to any one of claims 1 to 9, further comprising an encoding device, capable of encoding a microcarrier signal onto a magnetic microcarrier when trapped in said electrically and magnetically defined cage.

11. The device of claim 10, wherein said encoding device is a high spatial resolution light source, capable of bleaching a bleachable substrate.

12. A method for the identification and/or encoding of magnetic microcarriers, which comprises positioning and orienting said microcarriers in a liquid in an electrically and magnetically defined cage by application of an electrical and a magnetic field, so as to ensure a specific position and orientation of said microcarriers for said identification and/or encoding, and identifying and/or encoding said magnetic microcarriers.

13. The method of claim 12, wherein said encoding is provided by bleaching a pattern on a bleachable substrate.

14. The method of claim 12, wherein said encoding is provided by a high spatial resolution light source.

15. The method according to any one of claims 12 to 14, wherein said identification is ensured by a detection means comprising a confocal microscope whose optical axis is placed perpendicular to said magnetic field.

16. The method according to any one of claims 12 to 15, further comprising the step of sorting said magnetic microcarriers after said identification and/or encoding.

17. A microfluidic container for use in the device of any one of claims 1 to 11 , comprising one or more magnetic microcarriers.