EP1608587B1 - Vorbefüllte vorrichtungen im mikromassstab - Google Patents

Vorbefüllte vorrichtungen im mikromassstab Download PDF

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Publication number
EP1608587B1
EP1608587B1 EP04722754.1A EP04722754A EP1608587B1 EP 1608587 B1 EP1608587 B1 EP 1608587B1 EP 04722754 A EP04722754 A EP 04722754A EP 1608587 B1 EP1608587 B1 EP 1608587B1
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Prior art keywords
solid phase
bed
microfluidic device
phase material
affinity
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French (fr)
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EP1608587A1 (de
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Mats INGANÄS
Helene Derand
Susanna Lindman
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Gyros Patent AB
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Gyros Patent AB
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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
    • B01L3/502746—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 the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • B01L2300/069—Absorbents; Gels to retain a 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B01L2300/0806—Standardised forms, e.g. compact disc [CD] format
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • 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
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • 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
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • 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
    • B01L3/502707—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 the manufacture of the container or its components

Definitions

  • the solid phase material is present in the device as porous beds during the experiments.
  • the device permits that one or more experiments can be carried out in parallel within the device.
  • Parallelity means that at least the interaction between the solute and the solid phase material is carried out in parallel for two or more experiments.
  • the reagents/reactants used may be different.
  • solute comprises true solutes, microorganisms including viruses, suspended cells, suspended cell parts and various other reactants that are in dissolved or colloidal form and sufficiently small to be transported by liquid flow through the porous bed that is referred to herein.
  • microfluidic device means that the device comprises one or more microchannel structures in which liquid flow is used for transporting various kinds of reactants, analytes, products, samples, buffers and/or the like.
  • micro in “microchannel structure” contemplates that there are one or more cavities and/or conduits that have a cross-sectional dimension that is ⁇ 10 3 ⁇ m, preferably ⁇ 5 x 10 2 ⁇ m, such as ⁇ 10 2 ⁇ m.
  • the device is capable of processing liquid aliquots in the nanolitre (nl) range (which includes the picolitre (p1) range).
  • the nl-range has an upper end of 5,000 nl but relates in most cases to volumes ⁇ 1,000 nl, such as ⁇ 500 nl or ⁇ 100 nl.
  • WO 02075312 (Gyros AB) focuses on affinity assays for the characterization of reaction variables by binding a soluble affinity reactant to a solid phase material that comprises in immobilized form the counterpart to the affinity reactant.
  • the solid phase is represented by the inner wall of the reaction microcavity or by a porous bed placed in the reaction microcavity.
  • WO 03093802 describes performing catalytic assays with one part of the used catalytic system in immobilized form.
  • the assays are illustrated with enzyme systems.
  • the immobilization techniques and solid phase materials are in principle the same as in WO 02075312 (Gyros AB).
  • US 5,726,026 (Univ. Pennsylvania) and US 5,928,880 (Univ. Pennsylvania) describe in a side sentence a microfluidic device that comprises a detection/reaction zone containing a solid phase material in particle form. Streptavidin is immobilized to the particles. The particles may be dried or lyophilized.
  • US 6,479,299 discusses predispensation of soluble and insoluble reagents (assay components) during the manufacture of a microfluidic device.
  • Insoluble reagents may be in lyophilized form.
  • a microscale fluidic device (Gyrolab MALDI SP1) containing a plurality of microchannel structures each of which contains a column of a reverse solid phase material (hydrophobic beads) ( WO 02075775 (Gyros AB) and WO 02075776 (Gyros AB)).
  • the solid phase material is in a dry state.
  • the packages of the devices have been specifically designed.
  • WO 00056808 (Gyros AB), WO 01047437 (Gyros AB), WO 01054810 (Gyros AB), WO 02075775 (Gyros AB) and WO 02075776 (Gyros AB) suggest in general terms to deliver microfluidic devices in dry form.
  • US 5,354,654 (Ligler et al ) suggests a kit comprising a solid support with an immobilized ligand-receptor complex that has been lyophilized together with a cryostabilisator. Packing of the support in a macroscale column is suggested.
  • compositions having a high biotin-binding activity are immobilized to a polymer support.
  • the support may be in beaded form and lyophilized together with (a) a bulking agent protecting the beads from damages during freeze-drying and assisting the reswelling of the beads, (b) a protectant for inhibiting chemical reactions during freeze-drying and storage, (c) buffers etc.
  • the objects are to provide improved microfluidic devices that solve the problems discussed above.
  • the objects thus comprise to provide microfluidic devices comprising solid phase material in a dry state that after storage and transportation of the device can be reconstituted to wet beds with essentially the same performance as wet beds of the same solid phase material not having being transformed to the dry state.
  • the solid phase material comprises an immobilized reactant
  • its activity e.g. binding activity such as capacity to bind the solute
  • the objects include providing methods for manufacturing the devices and use of the devices for separation and/or assay purposes, among others.
  • a compound or a combination of compounds that reduces/reduce these adverse effects will henceforth be called “bed-preserving agent” or simply “preserver” since they will assist in restoring a dried solid phase material to an efficient wet porous bed.
  • a bed-preserving agent is simply included in the liquid phase of a wet solid phase material before drying/dehydration. Drying can take place inside or outside the microfluidic device.
  • inlet arrangements 102,103a-h
  • 106a-h distribution manifold
  • 108a - h single volume metering units
  • This aspect is a microfluidic device that comprises one, two or more microchannel structures (101), each of which comprises a reaction microcavity (104a-h) intended for retaining a solid phase material in the form of a porous bed.
  • the device is characterized in that the reaction microcavity (104a-h) in one, two or more of the microchannel structures (101) comprises a hydrophilic solid phase material in a dry state that comprises a compound or a combination of compounds that act as a bed-preserving agent. These compounds thus secure that an acceptable wet porous bed can be restored after a reconstitution liquid has passed the dry state solid phase material.
  • the bed preserving agent(s) is(are) capable of
  • acceptable wet porous bed means that the experimental results from the bed can be used, i.e. the bed is functional.
  • unacceptable means that the experimental results are discarded.
  • the bed-preserving agent thus increases the probability for obtaining acceptable beds.
  • the use of the principles of the invention may thus assist in increasing the yield of functional beds or microchannel structures on a microfluidic device to become ⁇ 70 %, such ⁇ 80 % or ⁇ 90% or ⁇ 95% or ⁇ 98 % of the total number of beds or microchannel structures of a microfluidic device.
  • dry state is meant that the amount of remaining liquid after drying is ⁇ 50 %, such as ⁇ 30 % or ⁇ 20 % or ⁇ 10 % of the amount of liquid present in the solid phase material when saturated with the liquid concerned (with no free liquid layer appearing on top of the bed). In many cases this means that the amount of liquid in the solid phase material after drying and/or storage is ⁇ 20 % (w/w), such as ⁇ 10 % or ⁇ 5 %.
  • the liquid referred to is typically water.
  • the damages of a porous bed during drying/dehydration and storage typically depend on stresses induced during transformation from a wet state to a dry state in the similar manner as for biologically active material.
  • the choice of bed-preserving agent will depend on the conditions for drying, the solid phase material, kind of immobilized reactant etc.
  • the same compound(s) may act as bed-preserving agent for one solid phase material and/or immobilized reactant but negatively affect other combinations. It will thus be extremely important to test individual preserver candidates [either as single compounds or as combination(s) of compounds] and conditions for the transformation to the dry state and/or the conditions for storage and/or reconstitution before a candidate is used for a particular solid phase material. Testing is typically by trial and error and may include
  • Determination of the activity of the immobilized reactant/ligand may include determination of i) the activity profile in the flow direction and/or perpendicular to the flow direction (i.e. the distribution of activity in the bed), ii) the total activity of the bed etc, for instance by testing the bed behavior in a standard type of assay or in an actual future use of the porous bed.
  • the immobilized reactant is an affinity reactant that is able to capture a solute
  • the distribution of the solute in the bed after adsorption (capture) may be used to find abnormal local behavior caused by channels, cavities, air inclusions or local inactivation of the reactant, for instance.
  • the total amount of adsorbed solute may give a total view, e.g.
  • Adsorption in the context of testing is preferably performed under flow conditions, i.e. a liquid containing the solute is allowed to flow through the porous bed.
  • flow conditions i.e. a liquid containing the solute is allowed to flow through the porous bed.
  • the substeps during which the risk for damages is most significant are primarily the drying step (dehydration step) and the storage as such.
  • the freezing step may cause significant damages.
  • particular stabilisators may be required for each substep.
  • stabilizators have been termed according to kind of substep during which they are active, e.g. cryostabilisators refer to freezing, lyostabilisators to dehydration/drying and long term stabilisators to storage. See for instance Arakawa et al (Advanced Drug Delivery Reviews 46 (2001) 307-326 ).
  • the analogous categorization is used for bed-preserving agents.
  • bed-reconstitution agents Compounds that assist in the reconstitution of the dry solid phase material to the wet porous bed are called bed-reconstitution agents and are also bed-preserving agents.
  • a bed-preserving agent may be active in relation to at least one up to all of the steps: drying/dehydration, freezing, storage and reconstitution.
  • the efficiency of a particular agent will depend on the conditions for the particular step, solid phase material and/or immobilized reactant to be stabilized.
  • a bed-preserving agent that is useful in the present invention typically is hydrophilic in the sense that it is water-soluble. Many bed-preserving agents thus exhibit one or more heteroatoms selected from oxygen, nitrogen and sulphur, typically with a ratio between the total number of carbon atoms and the total number of oxygen, nitrogen and sulpur atoms which is ⁇ 6, such as ⁇ 4 or ⁇ 2.
  • Typical bed-preserving agents maybe found in the group consisting of compounds exhibiting a) carbohydrate structure which also includes sugar alcohol structure, b) polyhydroxy structure (i.e. organic polyols which also includes polyhydroxy polymers), c) amino acid structure including peptide structure and imino acid structure, d) inorganic salts, e) organic salts in particular carboxylates, f) amine structure including amino acid structure and ammonium structure, h) etc.
  • Suitable compounds with carbohydrate structures may be found amongst sucrose, lactose, glucose, trehalose, maltose, isomaltose, cellobiose, inositol, ethylene glycol, glycerol, sorbitol, xylitol, mannitol, polyethylene glycol possibly substituted in one or both of its end, dextran, maltodextrin, monosaccharides, disaccharides, polysaccharides including oligosaccharides etc.
  • Compounds with carbohydrate structures are typically also polyols.
  • Suitable polyols may be found amongst polyhydroxy polymers, such as polysaccharides, polyvinylalcohol possibly partially substituted on its hydroxy groups for instance with acetate or lower hydroxy alkyl groups (C 2-4 ), poly (lower hydroxy alkyl (C 2-4 ) acrylate) polymers and corresponding poly methacrylate polymers etc, and monomeric compounds having two or more hydroxy groups.
  • polyhydroxy polymers such as polysaccharides, polyvinylalcohol possibly partially substituted on its hydroxy groups for instance with acetate or lower hydroxy alkyl groups (C 2-4 ), poly (lower hydroxy alkyl (C 2-4 ) acrylate) polymers and corresponding poly methacrylate polymers etc, and monomeric compounds having two or more hydroxy groups.
  • each hydroxy group is attached directly to an sp 3 -hybridised carbon.
  • Suitable polymers are typically found amongst polymers that have a plurality of functional groups comprising a heteroatom selected from oxygen and nitrogen.
  • Relevant functional groups are -O(CH 2 CH 2 O) n - where n is ⁇ 2 such as ⁇ 5, amido such as -CONH- or -CONH 2 (where H may be replaced with a suitable hydrophilic organic group), hydroxy (OH), ester (-COOR, where R is a suitable hydrophilic organic group), etc.
  • Specific examples are polyethylene glycol, dextran and other polysaccharides, polyvinylpyrrolidone, polypeptides, the poly acrylate and methacrylate polymers mentioned above, the polyvinyl alcohols mentioned above etc.
  • polymer above also includes copolymer in which the specific polymer structure mentioned is a part
  • Bed-preserving agents that are lyostabilisators are believed to act during the drying/dehydration step by replacing water bound to the solid phase material to be stabilized. These bed-preserving agents thus primarily are found among compounds that may participate in hydrogen bonding/coordination with the solid phase material. With the present knowledge the most typical candidates for lyostabilization are found amongst polyols (including diols, triols etc), e.g. with a polymeric structure and/or carbohydrate structure (oligomeric is included in polymeric). In the case the solid phase material comprises an immobilised reactant, e.g.
  • bed-preserving agents such as lyostabilisators and stabilisators for long term storage are capable of existing in a glassy state in the reaction microcavity possibly in admixture with one or more of the other components that are present in the reaction microcavity.
  • the bed-preserving agents that are present in the dry state of a solid phase material are typically non-volatile. This does not exclude that volatile cryostabilisators are included during lyophilization.
  • the solid phase material that is in a dry state may also contain one or more so-called protectants that inhibit undesired chemical reactions of the solid phase material and/or the immobilized reactant. Suitable protectants are found amongst free radical scavengers, antioxidants, reducing agents etc.
  • the solid phase material in a dry state may also contain an appropriate buffer, such as a buffer with non-volatile buffering components, e.g. with at least one or two of the buffering components being anionic, such as in phosphate buffers, citrate buffers etc. Also other buffers may be used.
  • the buffering components typically provide an elevated buffer capacity within an appropriate pH interval of the range of pH 1-13 with preference for the range 3-11.
  • phosphate buffers in particular with potassium as counter-ion, are preferred.
  • additives such as one or more antimicrobial agents may also be included, e.g. a bacteriostat, a bacteriocid, a virucid etc.
  • a possible bulking agent may also be included as an additive.
  • the bulking agent may have bed-preserving effects on the solid phase material as discussed above for bed preserving agents in general.
  • Microcavity adherence agents (a kind of bed-preserving agents) cause the solid phase material to be retained in a reaction microcavity and therefore assist in restoring a dry state solid phase material to a wet porous bed. This kind of agents acts by causing particles to adhere to each other and/or to the inner walls of a reaction microcavity.
  • Microcavity adherence agents may be found amongst the bed-preserving candidates discussed above, for instance amongst those that exhibit carbohydrate and/or polymeric structure.
  • the various additives are typically present in the solid phase material that is in the dry state in an amount in the interval of 0.0001 - 25 %, such as ⁇ 0.001% or ⁇ 0.01% or ⁇ 0.1% and/or ⁇ 10 % or ⁇ 1 %. These intervals apply to each individual additive as well as to the total amount of additive with the proviso that the total amount should not exceed the upper limit of an interval.
  • the determination of optimal ranges of efficient amounts and sufficient bed-preserving effects of individual bed-preserving agents needs experimental testing as discussed above.
  • the %-figures refer to the weight of the additive(s) relative to the total weight of solid phase material in the dry state.
  • Additives are typically soluble in aqueous media so that they easily can be removed from the reconstituted porous bed, for instance by transporting liquid through the reconstituted wet bed (washing).
  • Reaction microcavity (104a-h) and the solid phase material were Reaction microcavity (104a-h) and the solid phase material.
  • the reaction microcavity (104a-h) is defined as the part of a microchannel structure (101a-h) in which the solid phase is present. This means that for solid phases in the form of porous beds, the bed volume and the reaction microcavity (104a-h) will coincide and have the same volume. If the solid phase is the inner wall of a microconduit, the reaction microcavity (104a-h) is defined as the volume between the most upstream and the most downstream end of the solid phase.
  • the reaction microcavity (104a-h) is typically a straight or bent microconduit that may or may not be continuously widening and/or narrowing. On the same device all reaction microcavities typically have essentially the same shape and/or size.
  • the reaction microcavities/microchannel structures (104a-h/101a-h) may be divided into groups where each group contains reaction microcavities that are not present in any of the other groups. Each group may be placed in a subarea of the device that is separate from the subareas of other groups.
  • the reaction microcavity (104a-h) has at least one cross-sectional dimension that is ⁇ 1,000 ⁇ m, such as ⁇ 500 ⁇ m or ⁇ 200 ⁇ m (depth and/or width).
  • the smallest cross-sectional dimension is typically ⁇ 5 ⁇ m such as ⁇ 25 ⁇ m or ⁇ 50 ⁇ m.
  • the total volume of the reaction microcavity is typically in the nl-range, such as ⁇ 5,000 nl, such as 1,000 nl or ⁇ 500 nl ⁇ 100 nl or ⁇ 50 nl or ⁇ 25 nl.
  • the porous bed is a) a population of porous or non-porous particles, or b) a porous monolith.
  • a monolithic bed may be in the form of a porous membrane or a porous plug.
  • porous particles have the same meaning as in WO 02075312 (Gyros AB).
  • Suitable particles are spherical or spheroidal (beaded) or non-spherical.
  • Suitable mean diameters for particles used as solid phases are typically found in the interval of 1-100 ⁇ m with preference for mean diameters that are ⁇ 5 ⁇ m, such as ⁇ 10 ⁇ m or ⁇ 15 ⁇ m and/or ⁇ 50 ⁇ m. Also smaller particles can be used, for instance with mean diameters down to 0.1 ⁇ m.
  • the design of outlet end (111a-h) of the reaction microcavity (104ah) and the particles should match each other so that the particles can be retained in the reaction microcavity (104a-h). Certain kinds of particles, in particular particles of colloidal dimension, may agglomerate.
  • the size of the agglomerate should be in the intervals given even if the agglomerating particles as such are smaller. See for instance WO 02075312 (Gyros AB). Diameters refer to the "hydrodynamic" diameters.
  • Particles to be used may be monodisperse (monosized) or polydisperse (polysized) in the same meaning as in WO 02075312 (Gyros AB).
  • the solid phase material may or may not be transparent.
  • the base material of a solid phase may be made of inorganic and/or organic material.
  • Typical inorganic materials comprise glass and typical organic materials comprise organic polymers.
  • Polymeric materials comprise inorganic polymers, such as glass and silicone rubber, and organic polymers that may be of synthetic or biological origin (biopolymers).
  • biopolymer includes semi-synthetic polymers in which there is a polymer backbone derived from a native biopolymer. Typical synthetic organic polymers are cross-linked and are often obtained by the polymerisation of monomers comprising polymerisable carbon-carbon double bonds.
  • Suitable monomers are hydroxy alkyl acrylates and corresponding methacrylates, acryl amides and methacrylamides, vinyl and styryl ethers, alkene substituted polyhydroxy polymers, styrene, etc.
  • Typical biopolymers may or may not be cross-linked. In most cases they exhibit a carbohydrate structure, e.g. agarose, dextran, starch etc.
  • hydrophilic in the context of a porous bed contemplates a sufficient wettability of the surfaces of the pores for water to be spread by capillarity all throughout the bed when in contact with excess water (absorption).
  • the expression also means that the inner surfaces of the bed that is in contact with water during the absorption shall expose a plurality of polar functional groups which each has a heteroatom selected amongst oxygen and nitrogen, for instance.
  • Appropriate functional groups can be selected amongst hydroxy groups, ethylene oxide groups (-X-[CH 2 CH 2 O-] n where n is an integer > 1 and X is nitrogen or oxygen), amino groups, amide groups, ester groups, carboxy groups, sulphone groups etc, with preference for those groups that are essentially uncharged independent of pH, for instance within the interval of 2-12.
  • ethylene oxide groups -X-[CH 2 CH 2 O-] n where n is an integer > 1 and X is nitrogen or oxygen
  • amino groups amino groups
  • amide groups amino groups
  • ester groups carboxy groups
  • sulphone groups etc
  • the hydrophilic functional groups may be present on or be a part of so called extender arms (tentacles).
  • the base material of a solid phase material is hydrophobic or not sufficiently hydrophilic, e.g. is based on a styrene (co)polymer
  • the surfaces that are to be in contact with an aqueous liquid may be hydrophilized.
  • Typical protocols comprise coating with a compound or mixture of compounds exhibiting polar functional groups of the same type as discussed above, treatment by an oxygen plasma etc.
  • the solid phase material in a dry state may be swellable when contacted with the reconstitution liquid.
  • Swellable materials are likely to be more prone to give problems related to (a) shrinkage/swelling, and inhomogeneous packing and/or through flow after reconstitution, and/or (b) escape of dry particles during storage and transportation.
  • the term "swellable" in this context means an increase in volume of the material (particles as such or a monolith) can be detected when the material in the dry state (as defined above) is contacted with the reconstitution liquid (that may be aqueous such as water).
  • the increase in volume may for instance be ⁇ 10 or ⁇ 75 % of the volume of the material in a dry state.
  • Solid phase materials that are not swellable according to this definition are considered non-swellable..
  • the solid phase material may be rigid or elastic.
  • the solid phase material may or may not contain an immobilized reactant that is capable of participating in an organic, an inorganic, a biochemical reaction etc with a solute.
  • an immobilized reactant that is capable of participating in an organic, an inorganic, a biochemical reaction etc with a solute.
  • the interaction between the immobilized reactant and the solute may be part of a separation process, a catalytic reaction, a solid phase synthesis, a solid phase derivatization etc.
  • Affinity bonds typically are based on: (a) electrostatic interactions, (b) hydrophobic interactions, (c) electron-donor acceptor interactions, and/or (d) bioaffinity binding.
  • Bioaffinity binding typically is complex and comprises a combination of interactions, such as (a)-(c) above.
  • An immobilized affinity counterpart (AC s ) may thus:
  • a bioaffinity reactant/ligand is a member of a bioaffinity pair.
  • Typical bioaffinity pairs are a) antigen/hapten and an antibody, b) complementary nucleic acids, c) immunoglobulin-binding protein and immunoglobulin (for instance IgG or an Fc-part thereof and protein A or G), d) lectin and the corresponding carbohydrate, e) biotin and (strept)avidin, e) members of an enzymatic system (enzyme-substrate, enzyme-cofactor, enzyme-inhibitor etc), f) an IMAC group and an amino acid sequence containing histidyl and/or cysteinyl and/or phosphorylated residues (i.e.
  • Antibody includes antigen binding fragments and mimetics of antibodies.
  • bioaffinity pair includes also affinity pairs in which one or both of the members are synthetic, for instance mimicking one or both of the members of a native bioaffinity pair.
  • IMAC stands for an immobilized metal chelate.
  • the immobilized reactant/ligand may also be a catalytic system or a member of a catalytic system, such as a catalyst, a cocatalyst, a cofactor, a substrate or cosubstrate, an inhibitor, a promotor etc.
  • a catalytic system also includes linked catalytic systems, for instance a series of systems in which the product of the first system is the substrate of the second catalytic system etc and whole biological cells or a part of such cells.
  • the immobilized affinity reactant (AC s ) should be selected to have the appropriate selectivity and specificity for interacting with the solute of interest to the solid phase material in relation to an intended application.
  • General methods and criteria for the proper selection of affinity reactants and reaction conditions are well known in the field.
  • the affinity constant is typically ⁇ 10 -8 mole/l or ⁇ 10 -9 mole/l.
  • This kind of assays typically includes that the solute is reacted with immobilized AC s under flow conditions and related to the amount of an analyte in an animal or biological sample (animal or biological sample include samples from mammals, such as human and other animal patients, and from experimental animals).
  • affinity constant may be relatively large, such as up to 10 -3 mole/l or up to 10 -4 mole/l or up to 10 -5 or up to 10 -7 mole/l, or relatively low, such as less than 10 -8 mole/ or less than 10 -11 mole/l.
  • the techniques for immobilization of a reactant/ligand may be selected amongst techniques that are commonly known in the field.
  • the linkage to the solid phase material may thus be via covalent bonds, affinity bonds (for instance biospecific affinity bonds), physical adsorption etc.
  • Immobilization via affinity bonds may utilize an immobilizing affinity pair in which one of the members (immobilized ligand or L) is firmly attached to the solid phase material, for instance covalently.
  • the other member (immobilizing binder, B) of the pair is used as a conjugate (immobilizing conjugate) comprising binder B and the affinity counterpart AC s to the solute S.
  • immobilizing affinity pairs are a) streptavidin/avidin/ neutravidin and a biotinylated reactant (or vice versa), b) antibody and a haptenylated reactant (or vice versa), c) an IMAC group and an amino acid sequence containing histidyl and/or cysteinyl and/or phosphorylated residues (i.e. an IMAC motif) linked to or being a part of a reactant, etc.
  • conjugates primarily refers to covalent conjugates, such as chemical conjugates and recombinantly produced conjugates (where both the moieties have peptide structure).
  • the immobilized ligand L has two or more binding sites for the immobilizing binder B, and/or the immobilizing binder B has one, two or more binding sites for the ligand L (or vice versa).
  • affinity constants that roughly are ⁇ 10 -13 mole/l, ⁇ 10 -12 mole/l, ⁇ 10 -11 mole/1 and ⁇ 10 -10 mole/l, respectively.
  • the preference is to select L and B amongst biotin-binding compounds and streptavidin-binding compounds, respectively, or vice versa.
  • the affinity constants discussed above refer to values obtained by a biosensor (surface plasmon resonance) from Biacore (Uppsala, Sweden), i.e. with the affinity reactant (AC s and L) immobilized to a dextran-coated gold surface.
  • At least one of the members of an affinity pair, in particular a bioaffinity pair, to be used in the present invention typically exhibits a structure selected amongst: a) amino acid structure including peptide structure such as poly and oligopeptide structure, b) carbohydrate structure, c) nucleotide structure including nucleic acid structure, d) lipid structure such as steroid structure, triglyceride structure etc.
  • affinity pair in this context refers to the immobilizing affinity pair (L and B), the immobilized affinity reactant and the solute (AC s and S) and other affinity pairs that may be used.
  • the solid phase material that is in a dry state may alternatively be in activated form.
  • the functional group that can be used on the desired reactant is typically selected amongst electrophilic and nucleophilic groups and depends on whether or not the activated group is nucleophilic or electrophilic, respectively.
  • Examples of functional groups that may be used are amino groups and other groups comprising substituted or unsubstituted -NH 2 , carboxy groups (-COOH/- COO - ), hydroxy groups, thiol groups, keto groups etc.
  • a microchannel structure (101a-h) of a microfluidic device comprises functional parts that permit the full protocol of an experiment to be performed within the structure.
  • a microchannel structure (101a-h) of the microfluidic device thus may comprise one, two, three or more functional parts selected among: a) inlet arrangement (102,103a-h) comprising for instance an inlet port/inlet opening (105a-b,107a-h), possibly together with a volume-metering unit (106a-h,108a-h), b) microconduits for liquid transport, c) reaction microcavity (104a-h); d) mixing microcavity/unit; e) unit for separating particulate matters from liquids (may be present in the inlet arrangement), f) unit for separating dissolved or suspended components in the sample from each other, for instance by capillary electrophoresis, chromatography and the like; g) detection microcavity; h) waste conduit/microcavity (112,115a-h);
  • a functional part may have more than one functionality, e.g. reaction microcavity (104a-h) and a detection microcavity may coincide.
  • Various kinds of functional units in microfluidic devices have been described by Gyros AB/Amersham Pharmacia Biotech AB: WO 99055827 , WO 99058245 , WO 02074438 , WO 02075312 , WO 03018198 ( US 20030044322 ), WO 03034598 , SE 03026507 ( SE 04000717 , US SN 60/508,508 ), SE 03015393 ( US SN 60/472,924 ) and by Tecan/Gamera Biosciences: WO 01087487 , WO 01087486 , WO 00079285 , WO 00078455 , WO 00069560 , WO 98007019 , WO 98053311 .
  • a reaction microcavity (104a-h) intended for a hydrophilic porous bed is connected to one or more inlet arrangements (upstream direction) (102,103a-h), each of which comprises an inlet port (105a-b,107a-h) and at least one volume-metering unit (106a-h,108a-h).
  • inlet arrangements upstream direction
  • 102,103a-h each of which comprises an inlet port (105a-b,107a-h) and at least one volume-metering unit (106a-h,108a-h).
  • the inlet arrangement (102) is common to all or a subset (100) of microchannel structures (101a-h) and reaction microcavities (104a-h) intended to contain the solid phase material and comprises a common inlet port (105a-b) and a distribution manifold with one volume-metering unit (106a-h) for each microchannel structure/reaction microcavity (101a-h/104a-h) of the subset (100).
  • each of the volume-metering units (106a-h,108a-h) in turn is communicating with downstream parts of its microchannel structure (101a-h), e.g. the reaction microcavity (104a-h).
  • Microchannel structures linked together by a common inlet arrangement (102) and/or common distribution manifold define a group or subset (100) of microchannel structures.
  • Each volume-metering unit (106a-h,108a-h) typically has a valve (109a-h,110a-h) at its outlet end.
  • This valve is typically passive, for instance utilizing a change in chemical surface characteristics at the outlet end, such as a boundary between a hydrophilic and hydrophobic surface (hydrophobic surface break) ( WO 99058245 (Amersham Pharmacia Biotech AB)) and/or in geometric/physical surface characteristics ( WO 98007019 (Gamera)).
  • the microfluidic device may also comprise other common microchannels/micro conduits connecting different microchannel structures. Common channels including their various parts such as inlet ports, outlet ports, vents, etc., are considered part of each of the microchannel structures they are communicating with.
  • microchannels make it possible to construe microfluidic devices in which the microchannel structures form networks. See for instance US 6,479,299 (Caliper).
  • Each microchannel structure has at least one inlet opening (105a-b,107a-h) for liquids and at least one outlet opening for excess of air (vents) (116a-i) and possibly also for liquids (circles in the waste channel (112)).
  • the microfluidic device may also comprise microchannel structures that have no reaction microcavity for retaining a solid phase material according to the invention.
  • the microfludic device contains a plurality of microchannel structures/device intended to contain the solid phase according to the invention.
  • Plurality in this context means two, three or more microchannel structures and typically is ⁇ 10, e.g. ⁇ 25 or ⁇ 90 or ⁇ 180 or ⁇ 270 or ⁇ 360.
  • the microcannel structures of a device maybe divided in groups or subsets (100), each of which may for instance be defined by the size and/or shape of the reaction microcavity, by a common microchannel (102,112), such as a common inlet arrangement (102) with manifold, common waste channel (112) etc.
  • the number of microchannel structures in a group or subset is typically in the interval 1-99 %, such as 5-50 % or 5-25 % or 10-50%, of the total number of microchannel structures of the device. This typically means that each group typically comprises from 3-15 or 3-25 or 3-50 microchannel structures. Each group may be located to a particular area of the device.
  • Inertia force may be used, for instance by spinning the disc as discussed in the subsequent paragraph.
  • Other useful forces are capillary forces, electrokinetic forces, non-electrokinetic forces such as capillary forces, hydrostatic pressure etc.
  • the microfluidic device typically is in the form of a disc.
  • the preferred formats have an axis of symmetry (C n ) that is perpendicular to or coincides with the disc plane, where n is an integer ⁇ 2, 3, 4 or 5, preferably ⁇ (C ⁇ ).
  • the disc may be rectangular, such as square-shaped and other polygonal forms but is preferably circular.
  • centrifugal force may be used for driving liquid flow.
  • Spinning the device around a spin axis that typically is perpendicular or parallel to the disc plane may create the necessary centrifugal force. In the most obvious variants at the priority date, the spin axis coincides with the above-mentioned axis of symmetry.
  • each microchannel structure comprises one upstream section that is at a shorter radial distance than a downstream section (from the spin axis).
  • the reaction microcavity intended for the porous bed is typically at a radial position intermediary to the two sections.
  • reaction microcavity is typically oriented with the flow direction radially outwards from the spin axis.
  • the preferred devices are typically disc-shaped with sizes and/or forms similar to the conventional CD-format, e.g. sizes that are in the interval from 10% up to 300 % of a circular disc with the conventional CD-radii (12 cm).
  • the upper and/or lower sides of the disc may or may not be planar.
  • Microchannels/microcavities of a microfluidic devices may be manufactured from an essentially planar substrate surface that exhibits the channels/cavities in uncovered form that in a subsequent step are covered by another essentially planar substrate (lid).
  • essentially planar substrate e.g. plastic polymeric material.
  • wettable hydrophilic
  • non-wettable hydrophobic
  • a surface has a water contact angle ⁇ 90° or ⁇ 90°, respectively.
  • inner surfaces of the individual parts should primarily be wettable, preferably with a contact angle ⁇ 60° such as ⁇ 50° or ⁇ 40° or ⁇ 30° or ⁇ 20°.
  • These wettability values apply for at least one, two, three or four of the inner walls of a microconduit. In case one or more of the inner walls have a higher water contact angle this can be compensated for by a lower water contact angle for the inner wall(s).
  • a hydrophilic inner surface in a microchannel structure may comprise one or more local hydrophobic surface breaks in a hydrophilic inner wall, for instance for introducing a passive valve, an anti-wicking means, a vent solely function as a vent to ambient atmosphere etc (rectangles in figure 1 ). See for instance WO 99058245 (Gyros AB) and WO 02074438 (Gyros AB).
  • Contact angles refer to values at the temperature of use, typically +25°C, are static and can be measured by the method illustrated in WO 00056808 (Gyros AB) and WO 01047637 (Gyros AB).
  • SECOND ASPECT METHOD FOR THE TRANSFORMATION OF A PLURALITY OF WET POROUS BEDS TO A DRY/DEHYDRATED STATE THAT POSSIBLY IS RECONSTITUTED TO A PLURALITY OF WET POROUS BEDS.
  • This aspect is a method as defined in the heading of this section.
  • the method is characterized in comprising the steps of:
  • This aspect also concerns a method for reducing the inter-channel variation in a microfluidic device with respect to performance of reconstituted porous beds.
  • the solid phase material may or may not exhibit a reactant that can interact with a solute in a subsequently introduced liquid aliquot. Various characteristics are discussed below and elsewhere in this specification.
  • Step (iii) is preferably carried out under flow conditions, for instance with residence time and flow rates through the bed as discussed for the third aspect of the invention.
  • Porous particle beds can be created by flowing a dispersion of particles through all or one or more subsets (100) of the reaction microcavities (104a-h) of the microfluidic device. The particles will then settle and form a porous bed at the outlet end (111a-h) of each microcavity (104a-h). Bed formation may be facilitated by the use of gravity and/or the use of centrifugal force, the latter preferably acting along the flow direction of each reaction microcavity (104a-h).
  • the desired additives as discussed above are present in the liquid dispersion and/or introduced by passing a liquid containing the additives through the bed after it has been formed.
  • the microfluidic device together with the beds saturated with a liquid containing the additives is saved until transformation to the dry state.
  • a porous monolithic bed is typically introduced during the manufacture of the device, for instance
  • the preferred variant is to carry out the polymerization with the reaction microcavity (104a-h) and the corresponding microchannel structure (101a-h) in an enclosed form.
  • the preferred variant is to insert the monolith while at least the reaction microcavity (104a-h) is uncovered (and remaining part of the microchannel structure (101a-h) is covered). After introduction of the porous bed and if needed enclosing the microcavity, the beds are saturated with a solution comprising the additives discussed above and saved until transformation to the dry state.
  • Transformation of the beds to the dry state may be accomplished by removing the liquid under subatmospheric pressure, for instance below and/or above the freezing point of the liquid they are saturated with. Removal under subatmospheric pressure and below the freezing point typically means lyophlization. Alternatively liquid is removed from the settled dispersion under the pressure of ambient atmosphere with or without warming.
  • spin-drying In the case the device is designed for driving liquid transport by centrifugal force so called spin-drying may be employed. See description of figure 1 in the experimental part.
  • the reconstitution of the wet porous beds means that a reconstitution liquid is allowed to flow through each of the reaction microcavities containing solid phase material in a dry state. See the experimental part.
  • this kind of design will facilitate parallel dispensation of solid phase material as well as parallel reconstitution and conditioning of porous beds.
  • the use of the innovative microfluidic devices comprises in general terms the steps of:
  • the solute (S') is typically capable of interacting with the wet porous bed.
  • Step (iii) comprises that the solute (S') is formed within the device/microchannel structure or is dispensed to the microchannnel structure. If applicable, formation is typically in a position upstream or within the wet porous bed/reaction microcavity (104a-h). Dispensing is typically to an inlet port (105a-b,107a-h) at a position upstream the porous bed/reaction microcavity (104a-h).
  • Steps (iii) and (iv) are performed in order to allow for an interaction between the solute (S') and the porous bed to take place.
  • the steps may be part of (a) a separation method, and/or (b) a catalytic reaction, (c) a solid phase synthesis, and/or (d) a derivatization of the solid phase material/porous bed.
  • the separation may be part of a purification or enrichment protocol for a solute that is present in the liquid.
  • the solute that is separated from the liquid may be a contaminant or the entity to be purified, enriched etc.
  • the separation may also be part of a synthetic protocol, preparative protocol, a cell based assay, various kinds of affinity assays including nucleic acid assays, immunoassays, enzyme assays etc.
  • An affinity assay utilizing a capturing step for binding a solute to a solid phase material typically contemplates characterization of a reaction variable involved in an affinity reaction of the assay.
  • Reaction variables in this context are mainly of two types: 1) variables related to affinity reactants, and 2) reaction conditions.
  • Variables of type 1 comprises two main subgroups a) amounts including presence and/or absence, concentration, relative amounts, activity such as binding activity and enzyme activity, etc, and b) properties of affinity reactants including affinity as such, e.g. affinity constants, specificities etc. See WO 02075312 (Gyros AB).
  • the molecular entity for which a reaction variable of type 1 is characterized is called an analyte.
  • Catalytic reactions in the context of the present invention comprises that the solid phase material exhibits one or more immobilized members (e.g. affinity structure, affinity ligand, affinity reactant) of the catalytic system utilized, while other members of the same system are solutes.
  • the catalytic reaction comprises formation of an affinity complex between the immobilized member (affinity structure, affinity ligand, affinity reactant) and at least one of the solute members. At least one of the members corresponds to the substrate for the catalytic system.
  • the reaction results in a product that typically has a different chemical composition and/or structure compared to the substrate. The product may or may not become immobilized to the bed during the reaction.
  • catalytic system includes single catalytic system and more complex variants comprising a series of linked single enzyme systems, whole cells, cell parts exhibiting enzymatic activity etc.
  • the bed may function as a catalytic reactor, such as an enzyme reactor.
  • the step during which interaction with the solute occurs may be part of a catalytic assay, such as an enzyme assay, for characterizing one or more members of the catalytic system or other reaction variables (e.g. reaction conditions).
  • the assay may be for determining the activity of a particular catalyst, substrate, co-substrate, cofactor, co-catalyst etc in a liquid sample.
  • the molecular entity/entities corresponding to the activity to be determined is/are called analyte/analytes. See for instance WO 03093802 (Gyros AB).
  • analyte includes the entity to be characterized in an original sample as well as analyte-derived entities formed during the assay and being related quantitatively to the analyte in the original sample.
  • the solute discussed above may be the original analyte or an analyte-derived entity.
  • Solid phase synthesis includes for instance polymer synthesis, such as oligopeptide and oligonucleotide synthesis and synthesis of other small molecules on a solid phase material.
  • the immobilized reactant used in polymer synthesis may exhibit the structure of the corresponding monomer, such as nucleotide, carbohydrate, amino acid structure, and mimetics of these structures.
  • Synthesis of libraries of immobilized members of combinatorial libraries is also included. Such members have relatively low molecular weights (e.g. ⁇ 10,000 dalton including a possible spacer to a polymeric backbone).
  • Solid phase derivatizatizion in the context of the present invention in most instances has as the goal to introduce an immobilized reactant or an activated functional group on the wet porous bed.
  • Solid phase derivatization thus includes introduction of reactive structures or groups that permit immobilization of a desired reactant via covalent bonds or via affinity/adsorptive bonds.
  • the resulting porous bed can be used as discussed above for capturing/separating the solute S from a liquid containing the solute S.
  • the transport during step (iv) comprises that the liquid is continuously flowing through the porous bed or that the liquid transport is halted when the liquid is within the bed.
  • the interaction between a reactant immobilized on the bed and a solute can thus take place under flow condition or under static conditions, respectively.
  • the flow rate and/or residence time may for instance be adjusted such that the amount of solute (S) becoming bound to an affinity counterpart (ACs) immobilized to the solid phase will reflect the actual reaction rate or affinity between an immobilized affinity reactant, typically AC S , and a solute, typically solute S, with a minimum of perturbation by diffusion (non-diffusion limiting conditions).
  • This also applies to the present invention but does not exclude that for applications where the primary interest is the total amount of bound/captured solute, capturing under flow conditions utilizing either diffusion limiting or non-diffusion limiting conditions can be used.
  • the appropriate flow rate through the porous bed thus depends on a number of factors, e.g.
  • the flow rate should give a residence time of ⁇ 0.010 seconds such as ⁇ 0.050 sec or ⁇ 0.1 sec with an upper limit that typically is below 2 hours such as below 1 hour.
  • Illustrative flow rates are within 0.01-1000 nl/sec, such as 0.01-100 nl/sec and more typically 0.1 - 10 nl/sec. These flow rate intervals may be useful for bed volumes in the range of 1-200 nl, such as 1-50 nl or 1-25 nl.
  • Residence time refers to the time it takes for a liquid aliquot to be in contact with the solid phase/porous bed in the reaction microcavity. These intervals are also applicable to other uses of the innovative microfluidic devices including separation, catalytic assays, solid phase synthesis, solid phase derivatization etc.
  • the microfluidic device used for the experiments was circular and of the same dimension as a conventional CD (compact disc). This microfluidic device will further on be called CD.
  • the CD contained 14 groups (100) of 8 microchannel structures (101a-h) arranged in an annular zone around the center (spin axis) of the disc with a common waste channel (112) for each group close to the periphery.
  • a group (100) of 8 microchannel structures (101a-h) is shown in figure 1 and is similar to and function in the same manner as the group of microchannel structures illustrated in figures 1-2 in WO 02075312 (Gyros AB) and the corresponding figures in WO 03024548 ( US 20030054563 ) (Gyros AB) and WO 03024598 ( US 20030053934 ) (Gyros AB).
  • the dimensions are essentially of the same size as in these earlier patent applications.
  • Each subset (100) comprises eight microchannel structures (101a-h) with one common inlet arrangement (102), one separate inlet arrangement (103a-h) per microchannel structure, and one reaction microcavity (104a-h) per microchannel structure.
  • the common inlet arrangement comprises a) two common inlet ports (105a-b) that also will function as outlet ports for excess liquid, and b) one volume-metering unit (106a-h) for each microchannel structure (101a-h).
  • the volume-metering units (106a-h) will function as a distribution manifold for the downstream parts of the microchannel structures.
  • Each of the separate inlet arrangements (103a-h) is part of only one microchannel structure and comprises an inlet port (107a-h) and a volume-metering unit (108a-h). Between each volume-metering unit (106a-h, 108a-h) and their downstream parts, respectively, there is a valve function (109a-h, 110a-h), preferably passive.
  • a reaction microcavity (104a-h) of a microchannel structure (101a-h) is located downstream both the common inlet arrangement (102) and a separate inlet arrangement (103a-h) of a microchannel structure (101a-h).
  • each reaction microcavity ( 104a - h ) is in the downstream direction connected to an outlet microconduit (113a-h) that in figure 1 is illustrated as an outward bent and has an outlet end (114a-h) connected to a waste function (115a-h).
  • a waste function (115a-h).
  • Vents (116a-i, hydrophobic breaks) together with the valves (109a-h, hydrophobic breaks) define the volume of the liquid aliquots to be distributed downstream from each the volume-metering unit (106a-h).
  • capillarity By applying the appropriate volume of aqueous liquid to the inlet port of an inlet arrangement, capillarity will fill the volume-metering unit(s) connected to the inlet port with liquid. By spinning the disc around its center, liquid can be forced to pass the valve (109a-h,110a-h) between a volume-metering unit and downstream parts.
  • the immunoassay was performed in an automated system.
  • the system (Gyrolab Workstation, prototype 2 instrument equipped with a Laser Induced Fluorescence (LIF) module, Gyros AB, Uppsala, Sweden) was equipped with a CD-spinner, holder for microtiter plates (MTP) and a robotic arm with a holder for 10 capillaries connected to 5 syringe pumps, 2 and 2.
  • MTP microtiter plates
  • Two of the capillaries transferred all the reagents and buffers from a MTP to either of the two common inlet ports (105a-b) in the CD.
  • the other eight capillaries transferred individual samples from a MTP to the separate individual inlet ports (107a-h) in the CD.
  • the Gyrolab Workstation is a fully automated robotic system controlled by application-specific software.
  • An application specific method within the software controls the spinning of the CD at the precisely controlled speeds and thereby controls the movement of liquids through the microstructures as the application proceeds.
  • Special software was included in order to reduce background noise.
  • the solid phase bead material packed in the microstructures of the microfluidic device could be of either a porous or solid nature.
  • PS polystyrene
  • the beads were modified by passive adsorption of phenyl-dextran (PheDex) to create a hydrophilic surface and were subsequently covalently coupled with streptavidin (Immunopure Streptavidin, Pierce, Perbio Science UK Limited, Cheshire, United Kingdom) using CDAP chemistry ( Kohn & Wilchek, Biochem. Biophys. Res. Commun. 107 (1982), 878-884 ).
  • Superdex Peptide and Sepharose HP have also been covalently coupled with streptavidin using CDAP chemistry (without phenyl-dextran coating).
  • Streptavidin-biotin is a well-known bioaffinity pair.
  • the polystyrene particles are solid and non-swellable in the reconstitution liquid used.
  • Superdex Peptide and Sepharose HP are porous for many affinity reactants and swellable in the reconstitution liquids used.
  • the devices were stored for one month at +4°C after which the dry columns were rewetted/reconstituted once with 15 mM phosphate buffer (PBS), pH 7,4 containing 0.15 M NaCl, 0.02% NaN 3 and 0.01% Tween® 20 via the common distribution channel and spinning at the appropriate rate. Every addition of solution delivers 200 nl liquid to the individual column (104a-h). Finally the function of the reconstituted beds was tested in the immunoassay given below at four different analyte (myoglobin) concentrations and compared with the corresponding beds that had not been dried/dehydrated. The results are presented in figures 4-5 and show that it is more or less imperative to include a bed-preserving agent in order to reconstitute the dry/dehydrated solid phase material to an efficient wet porous bed.
  • the protein concentration of the monoclonal antimyoglobin 8E11.1 was 1-10 mg/ml and it was incubated in room temperature for 1 h with 3 ⁇ molar excess of the biotinylation reagent in 15 mM PBS with 0.15 M NaCl before it was gel filtrated through either a NAP-5 column (Amersham Biosciences, Uppsala, Sweden) or a Protein Desalting Spin Column (Pierce, # 89849-P, Perbio Science UK Limited, Cheshire, United Kingdom).
  • the complete assay was analyzed in the Laser Induced Fluorescence (LIF) detector module. See more WO 02075312 (Gyros AB), WO 03025548 and US 20030054563 (Gyros AB), and WO 03056517 and 10/331,399 (Gyros AB).
  • LIF Laser Induced Fluorescence
  • Table1 METHOD SPIN PROFILE Rewetting of bead columns Spin 1 2500 rpm 5s, 6000 rpm 10s Wash of beads Spin 2 1200 rpm 2s, 2500 rpm 0,5s, 4000 rpm 10s, 6500 rpm 16s Transfer of biotinylated antibody Spin flow 1 1200 rpm 2s, 2500 rpm 0,5s, from 1200-1500 rpm 45s, 2000 rpm 35s, 3000 rpm 30s, 4000 rpm 10s, 5000 rpm 5s, 6000 rpm 10s Wash of beads and CD-structure 1 Spin 3 1200 rpm 2s, 2500 rpm 1s, 4000 rpm 15s, 6000 rpm 18s Transfer of myoglobin samples Spin flow 2 1000 rpm 5s, 2500 rpm 0,5s, from 1200-1500 rpm 90s, 2000 rpm 70s, 3000 rpm

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Claims (19)

  1. Mikrofluidische Vorrichtung, umfassend eine, zwei oder mehrere Mikrokanalstrukturen (101a-h), die jeweils einen Reaktionsmikrohohlraum (104a-h) umfassen, der zum Aufnehmen eines Festphasenmaterials in Form eines nassen porösen Betts vorgesehen ist, wobei eine jede der einen, zwei oder mehreren Mikrokanalstrukturen (101a-h) das Festphasenmaterial in einem trockenen Zustand umfasst, dadurch gekennzeichnet, dass das Festphasenmaterial ein Bettkonservierungsmittel umfasst, das eine oder mehrere Verbindungen umfasst, die das Festphasenmaterial während des Übergangs des nassen Zustands des Festphasenmaterials in den trockenen Zustand stabilisieren und das Wiederherstellen des Festphasenmaterials in trockenem Zustand als ein nasses poröses Bett unterstützen können, dass wenigstens eine der Verbindungen ein Mikrohohlraumanhaftungsmittel ist, das das Festphasenmaterial in dem Reaktionsmikrohohlraum halten kann, und dass wenigstens eine oder mehrere der Verbindungen ein Polyol sind.
  2. Mikrofluidische Vorrichtung nach Anspruch 1, dadurch gekennzeichnet, dass wenigstens eine der einen oder mehreren Verbindungen a) eine hydrophile Gruppe aufweist, die nichtionisch sein kann, aber nicht sein muss, und b) wasserlöslich ist.
  3. Mikrofluidische Vorrichtung nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass wenigstens eine der einen oder mehreren Verbindungen eine Kohlenhydratstruktur aufweist, wie etwa eine Polysaccharidstruktur oder eine Oligosaccharidstruktur.
  4. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-3, dadurch gekennzeichnet, dass wenigstens eine der einen oder mehreren Verbindungen ein Disaccharid ist, vorzugsweise Trehalose.
  5. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-4, dadurch gekennzeichnet, dass der trockene Zustand einen nicht flüchtigen Puffer umfasst, z. B. einen Phosphatpuffer, möglicherweise mit Kaliumion als einem Gegenion.
  6. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-5, dadurch gekennzeichnet, dass der trockene Zustand innerhalb der mikrofluidischen Vorrichtung erzielt wird.
  7. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-6, dadurch gekennzeichnet, dass der trockene Zustand unter subatmosphärischem Druck aus dem porösen Bett, das mit einer wässrigen Flüssigkeit gesättigt ist, beispielsweise über oder unter dem Gefrierpunkt der Flüssigkeit, oder durch Trocknen des mit Wasser gesättigten porösen Betts in Umgebungstemperatur mit oder ohne Erwärmung erzielt wird.
  8. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-7, dadurch gekennzeichnet, dass a) das Festphasenmaterial in Form von porösen oder nicht porösen Partikeln vorliegt und b) das poröse Bett ein gepacktes Bett dieser Partikel ist.
  9. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-8, dadurch gekennzeichnet, dass das Festphasenmaterial quellfähig oder nicht quellfähig ist.
  10. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-9, dadurch gekennzeichnet, dass eine jede der einen, zwei oder mehr Mikrokanalstrukturen (101a-h) eine Einlassanordnung (102, 103a-h) mit einer Volumenmesseinheit (106a-h, 108a-h) umfasst, die mit dem Reaktionsmikrohohlraum (104a-h) verbunden ist.
  11. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-10, dadurch gekennzeichnet, dass die Vorrichtung zwei oder mehr Mikrokanalstrukturen (101a-h) umfasst, die in eine, zwei oder mehr Gruppen (100) von Mikrokanalstrukturen unterteilt sind, wobei jede Gruppe eine Einlassanordnung (102) umfasst, die a) allen Mikrokanalstrukturen der Gruppe (100) gemeinsam ist, und b) (i) einen gemeinsamen Einlassanschluss (105a-b) und (ii) für jede Mikrokanalstruktur (101a-h) der Gruppe eine Volumenmesseinheit (106a-h) umfasst, die in Stromaufwärtsrichtung mit dem gemeinsamen Einlassanschluss (105a-b) und in Stromabwärtsrichtung mit dem Reaktionsmikrohohlraum (104a-h) der Mikrokanalstruktur (101a-h) verbunden ist.
  12. Mikrofluidische Vorrichtung nach einem der Ansprüche 10-11, dadurch gekennzeichnet, dass die Innenwand der Volumenmesseinheiten (106a-h, 108a-h) ausreichend hydrophil ist, um durch Kapillarwirkung gefüllt zu werden, sobald eine wässrige Flüssigkeit in die Einheit gelangt ist, und b) ein Ventil (109a-h, 110a-h) an ihrem Auslass, beispielsweise ein passives Ventil.
  13. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-12, dadurch gekennzeichnet, dass jede Mikrokanalstruktur (101) dazu ausgelegt ist, mittels Zentrifugalkraft einen Flüssigkeitsstrom durch wenigstens einen Teil der Struktur zu treiben.
  14. Mikrofluidische Vorrichtung nach einem der Ansprüche 1-13, dadurch gekennzeichnet, dass das Festphasenmaterial ein immobilisiertes Reaktionsmittel umfasst, in der Regel ein immobilisiertes Affinitätsreaktionsmittel ACS zum Auffangen eines gelösten Stoffs mittels Affinität.
  15. Mikrofluidische Vorrichtung nach Anspruch 14, dadurch gekennzeichnet, dass das immobilisierte Reaktionsmittel ein immobilisierter Ligand L ist, der ein Teil eines immobilisierenden Affinitätspaars ist, das L und den Affinitätspartner B für L umfasst, und das für die Immobilisierung eines Konjugats B-ACS an dem porösen Bett vorgesehen ist, wobei ACS ein Affinitätspartner zu einem gelösten Stoff S ist.
  16. Mikrofluidische Vorrichtung nach Anspruch 15, dadurch gekennzeichnet, dass die Affinitätskonstante (KS--AC) zur Bildung des Komplexes (S--AC) zwischen dem gelösten Stoff (S) und dem Affinitätspartner (ACS) des gelösten Stoffs, d. h. (KS--AC) = [S] [ACS]/[S--ACS], maximal 10-6 mol/l beträgt.
  17. Mikrofluidische Vorrichtung nach Anspruch 16, dadurch gekennzeichnet, dass die Affinitätskonstante des immobilisierenden Affinitätspaars, d. h. KL'-B'=[L'] [B']/[L'-B'], maximal 103 mal größer als die entsprechende Affinitätskonstante für Streptavidin und Biotin ist, mit Präferenz dafür, dass das Affinitätspaar L' und B' jeweils aus biotinbindenden Verbindungen bzw. streptavidinbindenden Verbindungen oder umgekehrt ausgewählt ist.
  18. Mikrofluidische Vorrichtung nach Anspruch 17, dadurch gekennzeichnet, dass B eine oder mehrere Bindungsstellen für L aufweist und L zwei oder mehr Bindungsstellen für B aufweist, oder umgekehrt.
  19. Mikrofluidische Vorrichtung nach einem der Ansprüche 16-19, dadurch gekennzeichnet, dass wenigstens eins von S und ACS und/oder wenigstens eins von L, B, ACS und S eine Struktur umfasst, die ausgewählt ist aus Peptidstruktur einschließlich Poly-/Oligopeptid und Proteinstruktur, Kohlenhydratstruktur, Nukleotidstruktur einschließlich Nukleinsäurestruktur und Lipidstruktur.
EP04722754.1A 2003-03-23 2004-03-23 Vorbefüllte vorrichtungen im mikromassstab Expired - Lifetime EP1608587B1 (de)

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